Myc g-quadruplex stabilizing small molecules and their use

ABSTRACT

Methods for treating a tumor, such as a benign or malignant tumor, are disclosed herein. The methods include administering a therapeutically effective amount of a small molecule that selectively binds to and stabilizes G-quadruplex DNA in the promoter of the c-MYC gene to the subject. The methods are also of use to decrease the size and/or number of metastases. Compounds for use in the disclosed methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 16/835,102, filedMar. 30, 2020, which is a continuation of U.S. application Ser. No.16/218,341, filed Dec. 12, 2018, now U.S. Pat. No. 10,604,499, issuedMar. 31, 2020, which is a divisional of U.S. application Ser. No.15/541,676, filed Jul. 5, 2017, now U.S. Pat. No. 10,196,372, issuedFeb. 5, 2019, which is the U.S. National Stage of InternationalApplication No. PCT/US2016/012222, filed Jan. 5, 2016, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Application No. 62/099,938, filed Jan. 5,2015. Each of the prior applications is incorporated by reference hereinin its entirety.

FIELD OF THE DISCLOSURE

This relates to small molecule compounds that bind and stabilize theG-quadruplex DNA formation in the promoter of the c-MYC gene, andtherefore can be used to reduce expression of the c-MYC gene in cells.The small molecule compounds can be used, for example, in methods oftreating or inhibiting a tumor having increased c-MYC expression in asubject.

BACKGROUND

The oncogenic transcription factor c-Myc has a pleiotropic role in awide range of cell processes and is deregulated in some 70% of humancancers. However, targeting the c-Myc protein directly has proven to bedifficult due to a lack of well-defined pockets amenable to smallmolecule binding. An alternative approach for suppressing c-Myc levelsin the cell is through stabilization of the G-quadruplex DNA formation(G4) present in the promoter of the c-MYC gene. Expression of theproto-oncogene c-MYC is regulated by a 27 base pair (Pu27) sequencefound in the nuclease hypersensitive element III (1) region (NHEIII₁) ofthe c-MYC gene known to form a G4. Formation of the quadruplex in thissequence is believed to result in a “kink” in the DNA that prevents thepolymerase from continuing along its reading frame, ultimately resultingin downregulation of the c-MYC gene.

The use of small molecules to stabilize the G4 conformation andconsequently decrease c-MYC expression is an attractive therapeutic goalin cancers where c-Myc is overexpressed. Unfortunately, although somequadruplex stabilizing small molecules have been shown to reduce c-Mycexpression in cells, these agents may not be selective and activitycannot always be attributed to a c-MYC quadruplex-dependent mechanism ofaction. New classes of potent, selective quadruplex stabilizing agentsthat are active in tissue culture models would be of substantial utilityas reagents to study c-Myc biology as well as potential therapeutics.

SUMMARY

The identification and characterization of a new class of small moleculec-MYC G4 ligands that selectively bind to and stabilize the G4 in thec-MYC promoter region and silence c-Myc expression is described herein.The compounds are useful, for example, in methods of reducing orinhibiting c-MYC expression in cells (such as cancer cells), as well asin methods of treating or inhibiting a c-MYC expressing tumor in asubject.

In some embodiments, a method of decreasing c-MYC expression in a cellis provided. The method comprises contacting the cell with an effectiveamount of a compound, or a pharmaceutically acceptable salt or esterthereof, having a structure of:

wherein:

A is a 6 membered aryl ring;

C is a 6-membered aryl ring;

each R¹ is independently selected from hydrogen oroptionally-substituted lower alkyl;

a is 0, 1, or 2;

R² is selected from hydroxyl or halogen;

R⁸ and R⁹ together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring;

each R³ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl;

b is 0 to 4;

R⁴ is selected from halogen, lower haloalkyl, or optionally-substitutedlower alkyl;

each R⁵ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl;

d is 0, 1, or 2;

R⁶ is selected from methyl, trifluoromethyl, or phenyl;

Z is selected from nitrogen or oxygen; and

if Z is nitrogen, IV is selected from hydrogen, hydroxyl, halogen, lowerhaloalkyl, optionally-substituted lower alkyl, and if Z is oxygen, IV isnot present. In some such embodiments, a is 0, R² is hydroxyl, and/or R⁶is methyl. In some such embodiments, R⁴ is lower haloalkyl (such astrifluoromethyl) and/or d is 0. In some such embodiments, R⁸ and R⁹together with the linking nitrogen atom form optionally-substitutedpyrrolidine, optionally-substituted imidizolidine,optionally-substituted pryazolidine, optionally-substituted piperidine,optionally-substituted piperazine, or optionally-substituted azepane.For example, R⁸ and R⁹ together with the linking nitrogen atom formpyrrolidine, imidizolidine, pryazolidine, piperidine, piperazine, orazepane.

In some embodiments of the method of decreasing c-MYC expression in acell, the method can include contacting the cell with an effectiveamount of a compound, or a pharmaceutically acceptable salt or esterthereof, having a structure of one of Compounds 1, 3, 6, 10, 12-13,18-20, 22-23, 26, or 28-29 as disclosed herein. In some embodiments ofthe method of decreasing c-MYC expression in a cell, the method caninclude contacting the cell with an effective amount of a compound, or apharmaceutically acceptable salt or ester thereof, having a structure ofCompounds 23.

In additional embodiments, compounds for use in the disclosed methodsare provided. In some embodiments, a compound, or a pharmaceuticallyacceptable salt or ester thereof, is provided, having a structure ofFormula III:

wherein:

each R¹ is independently selected from hydrogen oroptionally-substituted lower alkyl;

R² is selected from hydroxyl or halogen;

R⁸ and R⁹ together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring;

each R³ is independently selected from halogen, lower haloalkyl, oroptionally-substituted lower alkyl;

b is 0 to 4;

R⁴ is trifluoromethyl;

each R⁵ is independently selected from halogen, lower haloalkyl, oroptionally-substituted lower alkyl;

d is 0, 1, or 2;

R⁶ is selected from methyl, trifluoromethyl, or phenyl;

Z is selected from nitrogen or oxygen; and

if Z is nitrogen, R⁷ is selected from hydrogen, hydroxyl, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl, and if Z is oxygen, R⁷is not present; and

with the proviso that the compound does not comprise the structure setforth as any one of structures 12-13, 17-20, or 36-43. In someembodiments, a compound, or a pharmaceutically acceptable salt or esterthereof, having a structure of one of compounds 23-29, or 34 isprovided. Pharmaceutical composition comprising a disclosed compound andat least one pharmaceutically acceptable additive are also provided.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor.

FIGS. 1A and 1B show a set of schematic drawings illustrating the c-MYCPu22 G-quadruplex and its formation. (A) Schematic drawing of the c-MYCPu22 G-quadruplex. In structure on right, each circle represents adifferent nucleotide (indicated as thymine, T, guanine, G, and adenine,A). The quadruplex-forming promoter sequence of the NHE III₁ region ofwild type c-MycPu27 (SEQ ID NO: 1) and variant c-MycPu22 (SEQ ID NO: 2)are shown. (B) Cartoon depicting reversible formation of G-quadruplexDNA in c-MYC promoter region that controls gene transcription.Stabilization of the quadruplex with a small molecule inhibitstranscription.

FIGS. 2A-2C show a set of diagrams and graphs illustratingidentification and testing of c-MycPu22 quadruplex DNA binding agents.(A) Cartoon depicting a small molecule microarray screen to identifycompounds that bind to c-MycPu22 quadruplex DNA. (B) Structure of acompound identified from the screen to selectively bind to c-MycPu22.Compound 1 increases the melting temperature of quadruplex DNA asmeasured by circular dichroism (average of 4 trials±standard deviation).(C) Surface plasmon resonance experiment to measure the dissociationconstant of Compound 1 binding to c-MycPu22. Shown are the sensorgram(left) and binding isotherm (right).

FIGS. 3A-3C show a pair of Western blots and a graph illustrating thatcompound 1 inhibits c-MycPu22 quadruplex function. (A) PCR stop assay.Compound 1 inhibits PCR amplification of a synthetic wildtypeoligonucleotide sequence (nucleotides 3-19 of SEQ ID NO: 2) capable offorming a G4, but not the mutant sequence (SEQ ID NO: 3) that cannotform a G4. (B) Exon specific qPCR assay with CA-46 Burkitt's Lymphomacell line. Exon 1 (in red) remains under control of the G4 whiletranscription from exon 2 is not under control of a G4. Cells weretreated with 10 μM 1 for the time indicated. Real time polymerase chainreaction was carried out, after which the observed threshold cycle (Ct)was measured and normalized to the value for cells treated with a DMSOcontrol. The data represents an average of four replicates. Error barsrepresent standard deviation. (C) Western blot demonstrating resistanceof CA-46 cell line to inhibition of c-Myc translation.

FIGS. 4A-4D show a set of graphs and a Western blot illustrating theeffects of Compound 1 on cell viability and c-Myc translation. (A)Dose-dependent effects of Compound 1 on myeloma cell viability at 72 h.(B) Time-dependent inhibition of c-Myc transcription in myeloma cellsafter treatment with 10 μM 1, as measured by qPCR. Data is average of 2replicates and error bars represent standard deviation. (C) Inhibitionof c-Myc protein translation with 10 μM Compound 1 is sustained overtime. (D) Effects on cell viability and c-Myc protein translation byCompound 1 across a panel of multiple myeloma cell lines. Also includedare the resistant CA46 Burkitt's Lymphoma cell line and peripheral bloodmononucleocytes.

FIGS. 5A-5C show a set of graphs and digital images illustrating thatCompound 1 causes cell-cycle arrest in myeloma cells. (A) Cycle analysisfor L363 cells treated with 10 μM Compound 1. Compound 1 inducessustained G1 arrest. (B) Compound 1 does not induce significantapoptosis at 10 μM after 72 h. (C) Compound 1 induces a senescent statein myeloma cells at 10 μM after 72 h.

FIGS. 6A-6C show a series of graphs illustrating the effect of Compound1 treatment on gene expression. (A) Treatment with 10 μM of Compound 1for various times reduces c-Myc expression of while minimally affectingexpression of other G-quadruplex containing genes, as evaluated usingNanostring assays. (B) Treatment with 5, 10 or 12.5 μM of Compound 1 for48 hours reduces c-Myc expression of while minimally affectingexpression of other G-quadruplex containing genes, as evaluated usingqPCR analysis (data are the average log 2 value for ΔΔCt of threereplicates). (C) A series of analogs of Compound 1 were tested for anyeffect on c-Myc expression or L363 cell viability. The expression levelsof the BCL2 (1), KRAS (2), HIF1A (3), VEGFA (4), Rbl (5), and c-MYC (6)genes is shown.

FIG. 7 illustrates potential decomposition pathways for Compound 1.

FIG. 8 shows the 12 hits from the screen that were chosen for follow-upstudies. Molecules with similar chemical scaffolds are grouped in boxes.

FIG. 9 shows a circular dichroism spectrum of c-MycPu22 quadruplex DNA(SEQ ID NO: 2). Observed maximum (262 nm) and minimum (244 nm)demonstrate formation of a properly folded parallel-strandedG-quadruplex structure.

FIG. 10 shows a general scheme for synthesis of analogs of Compound 1.

FIG. 11 illustrates exemplary substitutions to Compound 1 that can bemade to generate analogs that stabilize the G4 in the c-MYC promoter andinhibit c-Myc expression.

FIG. 12 is a set of graphs showing the blood plasma levels of Compound23 after a single IP (lower graph) or IV (upper graph) injection (19mg/kg) in female nude mice.

FIG. 13 shows a Western blot showing c-MYC expression levels in L363subcutaneous xenografts that were harvested from xenograft bearing mice2 or 8 hours after a single dose of Compound 23 at 19 mg/kg or 38 mg/kg(administered IP).

FIGS. 14A and 14B are a set of graphs illustrating the effect ofCompound 1 or Compound 23 treatment on gene expression. (A) Treatment ofL363 cells with 5 μM Compound 23 for 24 or 48 hours reduced c-Mycexpression while minimally affecting expression of other G-quadruplexcontaining genes as evaluated using qPCR analysis. (B) Treatment of L363cells with 10 μM BRACO-19, 10 μM Compound 1 or 7.5 μM Compound 23 for 48hours reduced c-Myc expression while minimally affecting expression ofother G-quadruplex containing genes as evaluated using qPCR. Theexpression levels of the BCL2 (1), KRAS (2), HIF1A (3), VEGFA (4), andc-MYC (5) genes is shown.

Sequences

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜4 kb), which wascreated on Apr. 30, 2021, which is incorporated by reference herein. Inthe accompanying sequence listing:

SEQ ID NO: 1 is the nucleic acid sequence of a c-MycPu27 G4

SEQ ID NO: 2 is the nucleic acid sequence of a c-MycPu22 G4

SEQ ID NO: 3 is the nucleic acid sequence of a mutant c-MycPu22 thatdoes not form a G4 structure.

SEQ ID NO: 4 is the nucleic acid sequence of an oligonucleotide primer.

DETAILED DESCRIPTION I. Terms

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

Acyl: A group having the structure —C(O)R, where R may be, for example,optionally substituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl. Lower acyl groups are those that contain one tosix carbon atoms.

Acyloxy: A group having the structure —OC(O)R—, where R may be, forexample, optionally substituted alkyl, optionally substituted aryl, oroptionally substituted heteroaryl. Lower acyloxy groups contain one tosix carbon atoms.

Administration: To provide or give to a subject an agent, for example, asmall molecule compound that selectively binds to G4 quadruplex DNA inthe c-MYC promoter, by any effective route. Exemplary routes ofadministration include, but are not limited to, oral, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

Co-administration or co-administering refers to administration of atleast two therapeutic compounds within the same general time period, anddoes not require administration at the same exact moment in time(although co-administration is inclusive of administering at the sameexact moment in time). Thus, co-administration may be on the same day oron different days, or in the same week or in different weeks. Thetherapeutic compounds disclosed herein may be included in the samecomposition or they may each individually be included in separatecompositions. In certain embodiments, the two compounds may beadministered during a time frame wherein their respective periods ofbiological activity overlap. Thus, the term includes sequential as wellas coextensive administration of two or more compounds.

“Administration of” and “administering a” compound should be understoodto mean providing a compound, a prodrug of a compound, or apharmaceutical composition as described herein. The compound orcomposition can be administered by another person to the subject (e.g.,intravenously) or it can be self-administered by the subject (e.g.,tablets).

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for decreasing or reducing tumor growth in a subject.Agents include effector molecules and detectable markers. In someembodiments, the agent is a chemotherapeutic agent. The skilled artisanwill understand that particular agents may be useful to achieve morethan one result; for example, an agent may be useful as both adetectable marker and a chemotherapeutic agent.

Aliphatic: A group including alkyl, alkenyl, alkynyl, halogenated alkyland cycloalkyl groups. A lower aliphatic group is a branched orunbranched aliphatic group having from 1 to 10 carbon atoms. Alkanediyl,cycloalkanediyl, aryldiyl, alkanearyldiyl: A divalent radical derivedfrom aliphatic, cycloaliphatic, aryl, and alkanearyl hydrocarbons.

Alkenyl: A cyclic, branched or straight chain group containing onlycarbon and hydrogen, and contains one or more double bonds that may ormay not be conjugated. Alkenyl groups may be unsubstituted orsubstituted. Lower alkenyl groups contain one to six carbon atoms.

Alkoxy: A straight, branched or cyclic hydrocarbon configuration andcombinations thereof, including from 1 to 20 carbon atoms, preferablyfrom 1 to 6 carbon atoms (referred to as a “lower alkoxy”), morepreferably from 1 to 4 carbon atoms, that include an oxygen atom at thepoint of attachment. An example of an “alkoxy group” is represented bythe formula —OR, where R can be an alkyl group, optionally substitutedwith an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl,alkoxy or heterocycloalkyl group. Suitable alkoxy groups includemethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy,tert-butoxy cyclopropoxy, cyclohexyloxy, and the like.

Alkoxycarbonyl: An alkoxy substituted carbonyl radical, —C(O)OR, whereinR represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl or similar moiety.

Alkyl: A branched or unbranched saturated hydrocarbon group of 1 to 24carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl,hexadecyl, eicosyl, tetracosyl and the like. A lower alkyl group is asaturated branched or unbranched hydrocarbon having from 1 to 6 carbonatoms. Preferred alkyl groups have 1 to 4 carbon atoms. Alkyl groups maybe substituted alkyls wherein one or more hydrogen atoms are substitutedwith a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl,aryl, alkenyl, or carboxyl. For example, a lower alkyl or (C₁-C₆)alkylcan be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,pentyl, 3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkylcan be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,2-cyclopentylethyl, or 2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy,ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy,3-pentoxy, or hexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or5-hexenyl; (C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

Alkynyl: A cyclic, branched or straight chain group containing onlycarbon and hydrogen, and unless otherwise mentioned typically containsone to twelve carbon atoms, and contains one or more triple bonds.Alkynyl groups may be unsubstituted or substituted. A lower alkynylgroup is one that contains one to six carbon atoms.

Amine or Amino: A group of the formula —NRR′, where R and R′ can be,independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example,an “alkylamino” or “alkylated amino” refers to —NRR′, wherein at leastone of R or R′ is an alkyl.

Aminoalkyl: An alkyl group as defined above where at least one hydrogenatom is replaced with an amino group (e.g, —CH₂—NH₂).

Aminocarbonyl: A group that, alone or in combination, includes an aminosubstituted carbonyl (carbamoyl) radical, wherein the amino radical mayoptionally be mono- or di-substituted, such as with alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl,aralkoxycarbonyl and the like. An aminocarbonyl group may be—N(R)—C(O)—R (wherein R is a substituted group or H). A suitableaminocarbonyl group is acetamido.

Amide or Amido: A group that is represented by the formula —C(O)NRR′,where R and R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group.

Analog: A molecule that differs in chemical structure from a parentcompound, for example a homolog (differing by an increment in thechemical structure or mass, such as a difference in the length of analkyl chain or the inclusion of one of more isotopes), a molecularfragment, a structure that differs by one or more functional groups, ora change in ionization. An analog is not necessarily synthesized fromthe parent compound. A derivative is a molecule derived from the basestructure.

Aralkyl: An alkyl group wherein an aryl group is substituted for ahydrogen of the alkyl group. An example of an aralkyl group is a benzylgroup.

Aryl: A monovalent unsaturated aromatic carbocyclic group having asingle ring (e.g., phenyl) or multiple condensed rings (e.g., naphthylor anthryl), which can optionally be unsubstituted or substituted. Aheteroaryl group is an aromatic group that has at least one heteroatomincorporated within the ring of the aromatic group. Examples ofheteroatoms include, but are not limited to, nitrogen, oxygen, sulfur,and phosphorous. Heteroaryl includes, but is not limited to, pyridinyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl,and the like. The aryl or heteroaryl group can be substituted with oneor more groups including, but not limited to, alkyl, alkynyl, alkenyl,aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylicacid, or alkoxy, or the aryl or heteroaryl group can be unsubstituted.

Aryloxy or Heteroaryloxy: A group of the formula —OAr, wherein Ar is anaryl group or a heteroaryl group, respectively.

Cancer: A malignant tumor that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, thyroidcancer is a malignant tumor that arises in or from thyroid tissue, andbreast cancer is a malignant tumor that arises in or from breast tissue(such as a ductal carcinoma). Residual cancer is cancer that remains ina subject after any form of treatment given to the subject to reduce oreradicate the cancer. Metastatic cancer is a tumor at one or more sitesin the body other than the site of origin of the original (primary)cancer from which the metastatic cancer is derived. Cancer includes, butis not limited to, solid tumors.

Carboxylate or Carboxyl: The group —COO or —COOH. The carboxyl group canform a carboxylic acid. A substituted carboxyl is a —COOR group where Ris alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenatedalkyl, or heterocycloalkyl group. For example, a substituted carboxylgroup could be a carboxylic acid ester or a salt thereof (e.g., acarboxylate).

Chemotherapeutic agent: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Forexample, chemotherapeutic agents are useful for the treatment ofneuroblastoma. Particular examples of additional therapeutic agents thatcan be used include microtubule binding agents, DNA intercalators orcross-linkers, DNA synthesis inhibitors, DNA and RNA transcriptioninhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, andangiogenesis inhibitors. In one embodiment, a chemotherapeutic agent isa radioactive compound. One of skill in the art can readily identify achemotherapeutic agent of use (see for example, Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide toChemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S.,Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, CancerChemotherapy and Biotherapy: Principles and Practice (4th ed.).Philadelphia: Lippincott Willians & Wilkins, 2005; Skeel, Handbook ofCancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003).Combination chemotherapy is the administration of more than one agent totreat cancer.

Control: A sample or standard used for comparison with an experimentalsample. In some embodiments, the control is a sample obtained from ahealthy patient or a non-tumor tissue sample obtained from a patientdiagnosed with cancer. In other embodiments, the control is a tumortissue sample obtained from a patient diagnosed with cancer. In someembodiments, the control is a tumor tissue sample obtained from apatient diagnosed with cancer, where the patient has not receivedtreatment with a G4 stabilizing agent as disclosed herein. In stillother embodiments, the control is a historical control or standardreference value or range of values (such as a previously tested controlsample, such as a group of cancer patients with known prognosis oroutcome, or group of samples that represent baseline or normal values,such as the expression level of the c-MYC gene in a non-tumor tissue).

Cycloalkyl: A non-aromatic carbon-based ring composed of at least threecarbon atoms. Examples of cycloalkyl groups include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Aheterocycloalkyl group is a cycloalkyl group as defined above where atleast one of the carbon atoms of the ring is substituted with aheteroatom such as, but not limited to, nitrogen, oxygen, sulfur, orphosphorous. Cycloalkyl and heterocycloalkyl groups can be mono-cyclicor bi-cyclic.

Decrease or Reduce: To reduce the quality, amount, or strength ofsomething; for example a reduction in tumor burden. In one example, atherapy reduces a tumor (such as the size of a tumor, the number oftumors, the metastasis of a tumor, or combinations thereof), or one ormore symptoms associated with a tumor, for example as compared to theresponse in the absence of the therapy. In a particular example, atherapy decreases the size of a tumor, the number of tumors, themetastasis of a tumor, or combinations thereof, subsequent to thetherapy, such as a decrease of at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, orat least 90%. Such decreases can be measured using the methods disclosedherein.

Determining or detecting the level of expression of a gene product:Detection of a level of expression in either a qualitative orquantitative manner, for example by detecting nucleic acid molecules orproteins, for instance using routine methods known in the art.

Diagnosis: The process of identifying a disease by its signs, symptomsand results of various tests. The conclusion reached through thatprocess is also called “a diagnosis.” Forms of testing commonlyperformed include blood tests, medical imaging, urinalysis, and biopsy.

Ester: A carboxyl group-containing moiety having the hydrogen replacedwith, for example, a C₁₋₆alkyl group (“carboxyl C₁₋₆alkyl” or“alkylester”), an aryl or aralkyl group (“arylester” or “aralkylester”)and so on. CO₂C₁₋₃alkyl groups are preferred, such as for example,methylester (CO₂Me), ethylester (CO₂Et) and propylester (CO₂Pr) andincludes reverse esters thereof (e.g. —OCOMe, —OCOEt and —OCOPr).

Halogenated alkyl or Haloalkyl group: An alkyl group with one or morehydrogen atoms present on these groups substituted with a halogen (F,Cl, Br, I).

Hydroxyl: A group represented by the formula —OH.

Hydroxyalkyl: An alkyl group that has at least one hydrogen atomsubstituted with a hydroxyl group. A alkoxyalkyl group is an alkyl groupthat has at least one hydrogen atom substituted with an alkoxy groupdescribed above.

Isolated or Purified: An biological component is a component that hasbeen substantially separated or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, i.e., other chromosomal and extra-chromosomal DNA and RNA,proteins, lipids, and organelles. “Isolated” does not require absolutepurity. For example, the desired isolated biological component mayrepresent at least 50%, particularly at least about 75%, moreparticularly at least about 90%, and most particularly at least about98%, of the total content of the preparation. Isolated biologicalcomponents as described herein can be isolated by many methods such assalt fractionation, phenol extraction, precipitation with organicsolvents (for example, hexadecyltrimethylammonium bromide or ethanol),affinity chromatography, ion-exchange chromatography, hydrophobicchromatography, high performance liquid chromatography, gel filtration,iso-electric focusing, physical separation (e.g., centrifugation orstirring), and the like.

The term purified does not require absolute purity; rather, it isintended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Forexample, a compound preparation is purified such that the desiredpolysaccharide protein conjugate represents at least 50%, moreparticularly at least about 90%, and most particularly at least about98%, of the total content of the preparation.

Multiple myeloma: A malignancy of terminally differentiated antibodysecreting B cells with ˜20,000 new cases diagnosed yearly in the UnitedStates (Jemal et al., CA Cancer J Clin., 60:277-300, 2010). MM ischaracterized by the accumulation of clonal plasma cells in the bonemarrow (BM) and osteolytic bone lesions. The person of ordinary skill isfamiliar with tests used to determine the presence and severity of MM.For example, the Durie-Salmon staging system divides MM patients intothree stages: Stages I, II, and III, corresponding to low, intermediate,and high cell mass, depending upon the severity of anemia, calciumlevel, kidney function, presence or absence of bone lesions, and thequantity of abnormal proteins. Approximately 25 percent of people withMM have high-risk disease. Treatment options include chemotherapy,treatment with immune modulating medications, and Autologous Stem CellTransplant (ASCT) (Attal et al., N. Engl. J. Med., 1996; 335:91-97;Barlogie et al., Blood, 1997; 89:789-793). However, patients invariablyrelapse, and MM remains a universal fatal disease. See, e.g., Rajkumarand Kyle, (eds), Treatment of Multiple Myeloma and Related Disorders,1^(st); Cambridge University Press, New York, 2006.

c-Myc: A transcription factor known to be overexpressed in several typesof cancer, including lymphoma and multiple myeloma. c-Myc protein isencoded by the c-MYC gene. Expression of the c-MYC gene is regulated inpart by a 27 base pair (Pu27) sequence found in the nucleasehypersensitive element III(1) region (NHEIII₁) of the c-MYC promoterthat can form a G-quadruplex (G4) structure. Formation of the G4structure in this sequence is believed to result in a “kink” in the DNAthat prevents the polymerase from continuing along its reading frame,ultimately resulting in downregulation of the c-MYC gene.

N-heterocyclic: Mono or bicyclic rings or ring systems that include atleast one nitrogen heteroatom. The rings or ring systems generallyinclude 1 to 9 carbon atoms in addition to the heteroatom(s) and may besaturated, unsaturated or aromatic (including pseudoaromatic). The term“pseudoaromatic” refers to a ring system which is not strictly aromatic,but which is stabilized by means of delocalization of electrons andbehaves in a similar manner to aromatic rings. Aromatic includespseudoaromatic ring systems, such as pyrrolyl rings.

Examples of 5-membered monocyclic N-heterocycles include pyrrolyl,H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including1,2,3 and 1,2,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl,isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls),tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), anddithiazolyl. Examples of 6-membered monocyclic N-heterocycles includepyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, and triazinyl. The heterocycles may beoptionally substituted with a broad range of substituents, andpreferably with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl,halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono ordi(C₁₋₆alkyl)amino. The N-heterocyclic group may be fused to acarbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl,and anthracenyl.

Examples of 8, 9 and 10-membered bicyclic heterocycles include 1Hthieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl,benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl,indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl, and the like.These heterocycles may be optionally substituted, for example with C₁₋₆alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, hydroxy, mercapto,trifluoromethyl, amino, cyano or mono or di(C₁₋₆alkyl)amino Unlessotherwise defined optionally substituted N-heterocyclics includespyridinium salts and the N-oxide form of suitable ring nitrogens.

Pharmaceutical composition: A composition including an amount (forexample, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to induce the desired tumorresponse. It may also be accompanied by medications for its use fortreatment purposes. The unit dosage form may be, for example, in asealed vial that contains sterile contents or a syringe for injectioninto a subject, or lyophilized for subsequent solubilization andadministration or in a solid or controlled release dosage.

Pharmaceutically acceptable salt or ester: Salts or esters prepared byconventional means that include salts, e.g., of inorganic and organicacids, including but not limited to hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid,lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid,benzoic acid, phenylacetic acid, mandelic acid and the like.

Pharmaceutically acceptable salts of the presently disclosed compoundsalso include those formed from cations such as sodium, potassium,aluminum, calcium, lithium, magnesium, zinc, and from bases such asammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine, diethylamine,piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammoniumhydroxide.

These salts may be prepared by standard procedures, for example byreacting the free acid with a suitable organic or inorganic base. Anychemical compound recited in this specification may alternatively beadministered as a pharmaceutically acceptable salt thereof.“Pharmaceutically acceptable salts” are also inclusive of the free acid,base, and zwitterionic forms. Descriptions of suitable pharmaceuticallyacceptable salts can be found in Handbook of Pharmaceutical Salts,Properties, Selection and Use, Wiley VCH (2002). When compoundsdisclosed herein include an acidic function such as a carboxy group,then suitable pharmaceutically acceptable cation pairs for the carboxygroup are well known to those skilled in the art and include alkaline,alkaline earth, ammonium, quaternary ammonium cations and the like. Suchsalts are known to those of skill in the art. For additional examples ofpharmacologically acceptable salts, see Berge et al., J. Pharm. Sci.66:1 (1977).

Pharmaceutically acceptable esters include those derived from compoundsdescribed herein that are modified to include a carboxyl group. An invivo hydrolysable ester is an ester, which is hydrolysed in the human oranimal body to produce the parent acid or alcohol. Representative estersthus include carboxylic acid esters in which the non-carbonyl moiety ofthe carboxylic acid portion of the ester grouping is selected fromstraight or branched chain alkyl (for example, methyl, n-propyl,t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term addition salt as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like. The term “quaternaryamine” as used hereinbefore defines the quaternary ammonium salts whichthe compounds are able to form by reaction between a basic nitrogen of acompound and an appropriate quaternizing agent, such as, for example, anoptionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g.methyliodide or benzyliodide. Other reactants with good leaving groupsmay also be used, such as alkyl trifluoromethanesulfonates, alkylmethanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine hasa positively charged nitrogen. Pharmaceutically acceptable counterionsinclude chloro, bromo, iodo, trifluoroacetate and acetate. Thecounterion of choice can be introduced using ion exchange resins.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art. For a general discussion of prodrugs involving esters seeSvensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985).

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated.A variety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions that will notaffect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts, Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When anamine is deprotected, the resulting salt can readily be neutralized toyield the free amine. Similarly, when an acid moiety, such as aphosphonic acid moiety is unveiled, the compound may be isolated as theacid compound or as a salt thereof.

Phosphoryl: Moieties of the formula —P(O)OR—, wherein R may be H, analiphatic or aromatic moiety, a cation or a lone pair of electrons.Phosphoryl moieties may be further substituted to form phosphoramidates,phosphates and phosphonates.

Polyether moiety: An oligomer (which is inclusive of dimers and higherrepeating units) or a polymer. Illustrative polyether moieties includethose derived from an aliphatic polyether (e.g., paraformaldehyde,polyethylene glycol (PEG), polypropylene glycol, and polytetramethyleneglycol, and those derived from an aromatic polyether (e.g., polyphenylether or poly(p-phenylene oxide)). A preferred polyether moiety isderived from PEG, also referred to herein as a poly(ethylene oxide). ThePEG may be a straight chain PEG or a branched PEG. PEG is also inclusiveof methoxypolyethylene glycol. In certain embodiments, the number ofrepeating ethylene oxide units in the PEG moiety may range from 2 to 50,more particularly from 2 to 10. The polyether moiety may be covalentlybonded to the core motif via PEGylation procedures.

Small organic molecule: An organic molecule with a molecular weight ofabout 1000 daltons or less (for example about 900 daltons or less, about800 daltons or less, about 700 daltons or less, about 600 daltons orless, about 500 daltons or less, about 400 daltons or less, about 300daltons or less, about 200 daltons or less, or about 100 daltons orless). In some examples, a small organic molecule has a molecular weightof about 100-1000 daltons, about 200-900 daltons, about 300-700 daltons,about 200-500 daltons, or about 400-700 daltons.

Subject: Includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats. The term subject appliesregardless of the stage in the organism's life-cycle. Thus, the termsubject applies to an organism in utero or in ovo, depending on theorganism (that is, whether the organism is a mammal or a bird, such as adomesticated or wild fowl).

Substituted or Substitution: Replacement of a hydrogen atom of amolecule or an R-group with one or more additional R-groups. Unlessotherwise defined, the term “optionally-substituted” or “optionalsubstituent” as used herein refers to a group which may or may not befurther substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or3, more preferably 1 or 2 groups. The substituents may be selected, forexample, from C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₃₋₈cycloalkyl,hydroxyl, oxo, C₁₋₆alkoxy, aryloxy, C₁₋₆alkoxyaryl, halo, C₁₋₆alkylhalo(such as CF₃ and CHF₂), C₁₋₆alkoxyhalo (such as OCF₃ and OCHF₂),carboxyl, esters, cyano, nitro, amino, substituted amino, disubstitutedamino, acyl, ketones, amides, aminoacyl, substituted amides,disubstituted amides, thiol, alkylthio, thioxo, sulfates, sulfonates,sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl,sulfonylamides, substituted sulfonamides, disubstituted sulfonamides,aryl, arC₁₋₆alkyl, heterocyclyl and heteroaryl wherein each alkyl,alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groupscontaining them may be further optionally substituted. Optionalsubstituents in the case N-heterocycles may also include but are notlimited to C₁₋₆alkyl i.e. N—C₁₋₃alkyl, more preferably methylparticularly N-methyl.

Sulfinyl: The group —S(═O)H. A substituted sulfinyl or sulfoxide is asulfinyl group having the hydrogen replaced with, for example aC₁₋₆alkyl group (“C₁₋₆alkylsulfinyl” or “C₁₋₆alkylsulfoxide”), an aryl(“arylsulfinyl”), an aralkyl (“aralkyl sulfinyl”) and so on.C₁₋₃alkylsulfinyl groups are preferred, such as for example, —SOmethyl,—SOethyl and —SOpropyl.

Sulfonyl: The group —SO₂H. The sulfonyl group can be further substitutedwith a variety of groups to form, for example, sulfonic acids,sulfonamides, sulfonate esters and sulfones. A substituted sulfonyl is asulfonyl group having the hydrogen replaced with, for example aC₁₋₆alkyl group (“sulfonylC₁₋₆alkyl”), an aryl (“arylsulfonyl”), anaralkyl (“aralkylsulfonyl”) and so on. SulfonylC₁₋₃alkyl groups arepreferred, such as for example, —SO₂Me, —SO₂Et and —SO₂Pr.

Sulfonylamido or sulfonamide: The group —SO₂NH₂.

Thiol: The group —SH. A substituted thiol is a thiol group having thehydrogen replaced with, for example a C₁₋₆alkyl group (“—S(C₁₋₆alkyl)”),an aryl (“—S(aryl)”), or an aralkyl (“—S(alkyl)(aryl)”) and so on.

Therapeutically effective amount: The amount of an agent that alone, ortogether with one or more additional agents, induces the desiredresponse, such as, for example treatment of a tumor in a subject.Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject.

In one example, a desired response is to decrease the size, volume, ornumber (such as metastases) of a tumor in a subject. For example, theagent or agents can decrease the size, volume, or number of tumors by adesired amount, for example by at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, atleast 90%, or at least 95% as compared to a response in the absence ofthe agent.

Several preparations disclosed herein are administered intherapeutically effective amounts. A therapeutically effective amount ofa disclosed compound that is administered to a human or veterinarysubject will vary depending upon a number of factors associated withthat subject, for example the overall health of the subject. Atherapeutically effective amount can be determined by varying the dosageand measuring the resulting therapeutic response, such as the regressionof a tumor. Therapeutically effective amounts also can be determinedthrough various in vitro, in vivo or in situ immunoassays. The disclosedagents can be administered in a single dose, or in several doses, asneeded to obtain the desired response. However, the therapeuticallyeffective amount of can be dependent on the source applied, the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

Treating or Inhibiting a Disease: A therapeutic intervention thatreduces a sign or symptom of a disease or pathological condition relatedto a disease (such as a tumor). Treatment can also induce remission orcure of a condition, such as a tumor. In particular examples, treatmentincludes preventing a tumor, for example by inhibiting the fulldevelopment of a tumor, such as preventing development of a metastasisor the development of a primary tumor. Prevention does not require atotal absence of a tumor.

Reducing a sign or symptom of a disease or pathological conditionrelated to a disease, refers to any observable beneficial effect of thetreatment. Reducing a sign or symptom associated with a tumor can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject (such as a subject having a tumor whichhas not yet metastasized), a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease(for example by prolonging the life of a subject having tumor), areduction in the number of relapses of the disease, an improvement inthe overall health or well-being of the subject, or by other parameterswell known in the art that are specific to the particular tumor. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Tumor: An abnormal growth of cells, which can be benign or malignant.Cancer is a malignant tumor, which is characterized by abnormal oruncontrolled cell growth. Other features often associated withmalignancy include metastasis, interference with the normal functioningof neighboring cells, release of cytokines or other secretory productsat abnormal levels and suppression or aggravation of inflammatory orimmunological response, invasion of surrounding or distant tissues ororgans, such as lymph nodes, etc. “Metastatic disease” refers to cancercells that have left the original tumor site and migrate to other partsof the body for example via the bloodstream or lymph system.

The amount of a tumor in an individual is the “tumor burden” which canbe measured as the number, volume, or weight of the tumor. A tumor thatdoes not metastasize is referred to as “benign.” A tumor that invadesthe surrounding tissue and/or can metastasize is referred to as“malignant.” Examples of hematological tumors include leukemias,including acute leukemias (such as 11q23-positive acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myelogenousleukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,medullary thyroid carcinoma, papillary thyroid carcinoma,pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma and retinoblastoma). In several examples, atumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

An “established” or “existing” tumor is an existing tumor that can bediscerned by diagnostic tests. In some embodiments, and establishedtumor can be palpated. In some embodiments, and “established tumor” isat least 500 mm³, such as at least 600 mm³, at least 700 mm³, or atleast 800 mm³ in size. In other embodiments, the tumor is at least 1 cmlong. With regard to a solid tumor, and established tumor generally hasan robust blood supply, and has induced Tregs and myeloid derivedsuppressor cells (MDSC).

A person of ordinary skill in the art would recognize that thedefinitions provided above are not intended to include impermissiblesubstitution patterns (e.g., methyl substituted with 5 different groups,and the like). Such impermissible substitution patterns are easilyrecognized by a person of ordinary skill in the art. Any functionalgroup disclosed herein and/or defined above can be substituted orunsubstituted, unless otherwise indicated herein. Unless otherwiseexplained, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. The singular terms “a,” “an,” and “the”include plural referents unless context clearly indicates otherwise. Theterm “comprises” means “includes.” Therefore, comprising “A” or “B”refers to including A, including B, or including both A and B. It isfurther to be understood that all base sizes or amino acid sizes, andall molecular weight or molecular mass values, given for nucleic acidsor polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described herein. In case ofconflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

II. Compounds

Disclosed therein are novel methods for using G4 stabilizing compounds,or a pharmaceutically acceptable salt or ester thereof, for thetreatment of, for example, cancer. The compounds can selectively bind tothe G4 in the c-MYC promoter, for example G4 DNA comprising the nucleicacid sequence set forth as TGAGGGTGGGTAGGGTGGGTAA, SEQ ID NO: 2. Inseveral embodiments, the compounds can be used to reduce expression ofthe c-MYC gene in a cell. The cell can be in vitro or in vivo. In anon-limiting example, the cell is a cancer cell, such as a multiplemyeloma cell.

In one embodiment, there is disclosed herein a compound, or apharmaceutically acceptable salt or ester thereof, having a structureof:

wherein:

A, B, and C are each independently selected from a 4 to 7 memberedcycloaliphatic, optionally-substituted heterocycloaliphatic,optionally-substituted aryl, or optionally-substituted heteroaryl;

each R¹ is independently selected from hydrogen oroptionally-substituted lower alkyl;

a is 0 to 2, such as 0 to 1, for example 0;

R² is selected from hydroxyl, halogen, lower haloalkyl, oroptionally-substituted lower alkoxy, such as hydroxyl;

X is optionally-substituted methyl, ethyl or propyl, such as methyl;

each R³ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl, for example, R³ can bemethyl and b can be 1;

b is 0 to 6, such as 0 to 1, for example 1;

each Y is independently selected from optionally-substituted loweralkyl, optionally-substituted amide, optionally-substituted sulfonamide,or optionally-substituted phosphoramide;

c is 0 to 3, such as 1 to 2, for example 1;

R⁴ is selected from halogen, lower haloalkyl, or optionally-substitutedlower alkyl, such as lower haloalkyl, for example trifluoromethyl;

each R⁵ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl;

d is 0 to 5, such as 0 to 1, for example 0;

R⁶ is selected from halogen, hydroxyl, lower haloalkyl, oroptionally-substituted lower alkyl, such as lower alkyl, for examplemethyl;

Z is selected from carbon, oxygen, nitrogen, or sulfur, such as oxygen;and

wherein if Z is carbon or nitrogen, R⁷ is selected from hydroxyl,halogen, lower haloalkyl, or optionally-substituted lower alkoxy, or isnot present.

In some embodiments of Formula I, X is methyl.

In some embodiments of Formula I, Y is amide. In some embodiments ofFormula I, Y can be selected from one of:

and c can be selected from 1 or 2, for example, Y can be

and c can be 1.

In some embodiments of Formula I, B is optionally-substitutedN-heterocyclic or N-heterocyclic. In some embodiments of Formula I, B isselected from optionally-substituted pyrrolidine, optionally-substitutedimidizolidine, optionally-substituted pryazolidine,optionally-substituted pyrrole, optionally-substituted diazole,optionally-substituted triazole, optionally-substituted piperidine,optionally-substituted pyridine, optionally-substituted diazine,optionally substituted triazine, optionally-substituted piperazine,optionally-substituted azepane, or optionally-substituted azepine. Insome embodiments of Formula I, R³ is hydrogen or methyl. For example, insome embodiments of Formula I, a combination of B and R³ can be selectedfrom one of the following:

In one embodiment, there is disclosed herein a compound, or apharmaceutically acceptable salt or ester thereof, having a structureof:

wherein:

A and C are each independently selected from a 5 or 6 memberedoptionally-substituted aryl, or optionally-substituted heteroaryl;

each R¹ is independently selected from hydrogen oroptionally-substituted lower alkyl, such as methyl;

a is 0 to 2, such as 0 to 1, for example 0;

R² is selected from hydroxyl, halogen, lower haloalkyl, oroptionally-substituted lower alkoxy, such as hydroxyl;

R⁸ and R⁹ together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring;

each R³ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl;

b is 0 to 6;

R⁴ is selected from halogen, lower haloalkyl, or optionally-substitutedlower alkyl;

each R⁵ is independently selected from hydrogen, halogen, lowerhaloalkyl, or optionally-substituted lower alkyl;

d is 0 to 5;

R⁶ is selected from halogen, hydroxyl, lower haloalkyl, oroptionally-substituted lower alkyl;

Z is selected from nitrogen or oxygen; and

wherein if Z is nitrogen, R⁷ is selected from hydrogen, hydroxyl,halogen, lower haloalkyl, or optionally-substituted lower alkoxy, and ifZ is oxygen, R⁷ is not present.

In some embodiments of Formula II, A is a 6 membered aryl ring; C is a6-membered aryl ring; each R¹ is independently selected from hydrogen oroptionally-substituted lower alkyl; a is 0, 1, or 2; R² is selected fromhydroxyl or halogen; R⁸ and R⁹ together with the linking nitrogen atomform a 4 to 7 membered optionally-substituted N-heterocyclic ring; eachR³ is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 4; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0, 1, or 2; R⁶ is selected frommethyl, trifluoromethyl, or phenyl; Z is selected from nitrogen oroxygen; and if Z is nitrogen, IV is selected from hydrogen, hydroxyl,halogen, lower haloalkyl, or optionally-substituted lower alkyl, and ifZ is oxygen, IV is not present.

In some embodiments of Formula I or Formula II, C can be 6-memberedoptionally-substituted aryl or optionally substituted heteroaryl. Insome embodiments of Formula I or Formula II, R⁴ can be lower haloalkyl,such as trifluoromethyl. In some embodiments of Formula I or Formula II,d can be 0. For example, in some embodiments, C can be a benzyl ornitrobenzyl ring, and R⁴ can be methyl or trifluoromethyl. For example,in some embodiments,

can be selected from one of:

In some embodiments of Formula II, Z is not oxygen, R² is not hydroxyl,R⁶ is not methyl, B is not a 5-, 6-, or 7-membered N-heterocycloalkylring, and/or the compound does not comprise the structure set forth asany one of structures 1-13, or 17-20.

In some embodiments of Formula I or Formula II, A is a 6-memberedoptionally-substituted aryl or optionally-substituted heteroaryl. Insome embodiments of Formula I or Formula II, each of a is 0. In someembodiments of Formula I or Formula II, R² is hydroxyl or halide. Forexample, in some embodiments of Formula I or Formula II,

can be selected from one of:

wherein Ha is halide, such as F or Cl.

In some embodiments, there is disclosed herein a compound, or apharmaceutically acceptable salt or ester thereof, having a structure ofany one of Formulas III-IX

With reference to Formula III, in some embodiments, each R¹ isindependently selected from hydrogen or optionally-substituted loweralkyl or is not present; R² is selected from hydroxyl or halogen; R⁸ andR⁹ together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring; each R³ is independentlyselected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 6; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0 to 2; and R⁶ is selected frommethyl, trifluoromethyl, or phenyl; Z is selected from nitrogen oroxygen; if Z is nitrogen, R⁷ is selected from hydrogen, hydroxyl,halogen, lower haloalkyl, or optionally-substituted lower alkyl, and ifZ is oxygen, R⁷ is not present.

With reference to Formula III, in some embodiments, each R¹ isindependently selected from hydrogen or optionally-substituted loweralkyl; R² is selected from hydroxyl or halogen; R⁸ and R⁹ together withthe linking nitrogen atom form a 4 to 7 membered optionally-substitutedN-heterocyclic ring; each R³ is independently selected from halogen,lower haloalkyl, or optionally-substituted lower alkyl; b is 0 to 4; R⁴is trifluoromethyl; each R⁵ is independently selected from halogen,lower haloalkyl, or optionally-substituted lower alkyl; d is 0, 1, or 2;R⁶ is selected from methyl, trifluoromethyl, or phenyl; Z is selectedfrom nitrogen or oxygen; and if Z is nitrogen, R⁷ is selected fromhydrogen, hydroxyl, halogen, lower haloalkyl, or optionally-substitutedlower alkyl, and if Z is oxygen, R⁷ is not present; and with the provisothat the compound does not comprise the structure set forth as any oneof structures 12-13, 17-20, or 36-43.

With reference to Formula IV, in some embodiments, R⁸ and R⁹ togetherwith the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring; each R³ is independentlyselected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 6; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0 to 2; and IV is selected fromhydrogen, hydroxyl, halogen, lower haloalkyl, or optionally-substitutedlower alkyl.

With reference to Formula V, in some embodiments, R⁸ and R⁹ togetherwith the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring; each R³ is independentlyselected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 6; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; and d is 0 to 2.

With reference to Formulas VI and VII, in some embodiments, R⁸ and R⁹together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring; each R³ is independentlyselected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 6; and R⁷ is selected fromhydrogen, hydroxyl, halogen, lower haloalkyl, or optionally-substitutedlower alkyl.

With reference to Formulas VIII and IX, in some embodiments, R⁸ and R⁹together with the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocyclic ring; each R³ is independentlyselected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; and b is 0 to 6.

In some embodiments of any one of Formulas I-V, R⁴ is trifluoromethyland d is 0. In some embodiments of any one of Formulas I-V, R⁴ istrifluoromethyl and d is 1. In some embodiments of any one of FormulasI-V, R⁴ is methyl and d is 0. In some embodiments of any one of FormulasII-V, R⁴ is methyl and d is 1. In some embodiments of any one ofFormulas II-IX, the compound does not comprise the structure set forthas any one of structures 12-13, 17-20, or 36-43.

In some embodiments of any one of Formulas II-IX, R⁸ and R⁹ togetherwith the linking nitrogen atom form optionally-substituted pyrrolidine,optionally-substituted imidizolidine, optionally-substitutedpryazolidine, optionally-substituted piperidine, optionally-substitutedpiperazine, optionally-substituted azepane. In some embodiments of anyone of Formulas II-IX, R³ is hydrogen or methyl. In some embodiments ofany one of Formulas II-IX,

can be selected from one of:

In some embodiments of any one of Formulas II-IX, R³ is hydrogen ormethyl. For example, in some embodiments of any one of Formulas II-IX,

can be selected from one of:

In some embodiments of Formula I, Formula II, or Formula III, R⁶ ismethyl. In some embodiments of Formula I, Formula II, or Formula III, Zis nitrogen.

In some embodiments, of Formula I or Formula II, the compound can beselected from one of compounds 1, 3, 6, 7, 12, 13, or 17-25.

In several embodiments, the disclosed compounds can selectively bind tothe G4 in the c-MYC promoter, for example G4 DNA comprising the nucleicacid sequence set forth as TGAGGGTGGGTAGGGTGGGTAA, SEQ ID NO: 2. Incertain embodiments, the compounds disclosed herein have an equilibriumdissociation constant (K_(d)) for G4 DNA having the sequence set forthas SEQ ID NO: 2 of no more than 5 μM, such as no more than 1 μM, no morethan 0.5 μM, no more than 100 nM, no more than 10 nM, or no more than 1nM or less.

III. Methods of Use

In several embodiments, a method is provided for reducing c-Mycexpression in a cell. The method includes contacting the cell with aneffective amount of a disclosed compound (such as any of the compoundsdisclosed in section II, or one of Compounds 36-43) or apharmaceutically acceptable salt or ester thereof. The compoundselectively binds to G4 quadruplex DNA in the promoter of the c-MYCgene, and stabilizes the G4 formation, thereby reducing expression ofthe c-MYC gene in the cell. The cell can be in vitro or in vivo. Inseveral embodiments, the expression of the c-MYC gene in the cell isreduced at least 50% (such as at least 75%, at least 80%, at least 90%,at least 95%, or at least 98%) compared to the expression of the c-MYCgene in a corresponding control cell. In some embodiments, decreasingexpression of c-Myc in the cell decreases growth and/or proliferation ofthe cell.

In additional embodiments, a therapeutically effective amount of adisclosed compound (such as any of the compounds disclosed in sectionII, or one of Compounds 36-43) or a pharmaceutically acceptable salt orester thereof can be administered to a subject to treat or inhibit atumor and/or a cancer in a subject. The subject can be selected fortreatment that has, is suspected of having or is at risk of developing atumor, such as a lymphoma or multiple myeloma. Subjects that can benefitfrom the disclosed methods include human and veterinary subjects. Insome embodiments, treating the tumor and/or cancer in the subjectdecreases growth and/or proliferation of the tumor.

The tumor can be benign or malignant. The tumor can be any tumor ofinterest, including, but not limited to lymphoma or multiple myeloma.Additional examples are skin tumors, breast tumors, brain tumors,cervical carcinomas, testicular carcinomas, head and neck tumors,gastrointestinal tract tumors, genitourinary system tumors,gynaecological system tumors, breast, endocrine system tumors, skintumors, a sarcoma of the soft tissue and bone, a mesothelioma, amelanoma, a neoplasm of the central nervous system, or a leukemia. Insome embodiments, the tumor is a head and neck tumor, such as tumors ofthe nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands and paragangliomas. Inother embodiments, the tumor is a lung tumor, such as a non-small celllung cancer or a small cell lung cancer. In further embodiments, thetumor can be a tumor of the gastrointestinal tract, such as cancer ofthe oesophagus, stomach, pancreas, liver, biliary tree, small intestine,colon, rectum and anal region. In yet other embodiments, the tumor canbe a tumor of the genitourinary system, such as cancer of the kidney,urethra, bladder, prostate, urethra, penis and testis. In someembodiments, the tumor is a gynecologic tumor, such as cancer of thecervix, vagina, vulva, uterine body, gestational trophoblastic diseases,ovarian, fallopian tube, peritoneal, or breast. In other embodiments,the tumor is an endocrine system tumor, such as a thyroid tumor,parathyroid tumor, adrenal cortex tumor, pancreatic endocrine tumor,carcinoid tumor and carcinoid syndrome. The tumor can be a sarcoma ofthe soft tissue and bone, a mesothelioma, a cancer of the skin, amelanoma, comprising cutaneous melanomas and intraocular melanomas, aneoplasm of the central nervous system, a cancer of the childhood,comprising retinoblastoma, Wilm's tumor, neurofibromatoses,neuroblastoma, Ewing's sarcoma family of tumors, rhabdomyosarcoma. Thetumor can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneousT-cell lymphomas, primary central nervous system lymphoma, and

Hodgkin's disease. The tumor can be a leukaemia, such as acuteleukemias, chronic myelogenous and lymphocytic leukemias. The tumor canbe plasma cell neoplasms, a cancer of unknown primary site, a peritonealcarcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas,AIDS-associated primary central nervous system lymphoma, AIDS-associatedHodgkin's disease and AIDS-associated anogenital cancers, a metastaticcancer to the liver, metastatic cancer to the bone, malignant pleuraland pericardial effusions and malignant ascites.

Treatment of the tumor is generally initiated after the diagnosis of thetumor, or after the initiation of a precursor condition (such asdysplasia or development of a benign tumor). Treatment can be initiatedat the early stages of cancer, for instance, can be initiated before asubject manifests symptoms of a condition, such as during a stage Idiagnosis or at the time dysplasia is diagnosed. However, treatment canbe initiated during any stage of the disease, such as but not limited tostage I, stage II, stage III and stage IV cancers. In some examples,treatment is administered to these subjects with a benign tumor that canconvert into a malignant or even metastatic tumor.

Treatment initiated after the development of a condition, such asmalignant cancer, may result in decreasing the severity of the symptomsof one of the conditions, or completely removing the symptoms, orreducing metastasis, tumor volume or number of tumors. In some example,the tumor becomes undetectable following treatment. In one aspect of thedisclosure, the formation of tumors, such as metastasis, is delayed,prevented or decreased. In another aspect, the size of the primary tumoris decreased. In a further aspect, a symptom of the tumor is decreased.In yet another aspect, tumor volume is decreased.

Subjects can be screened prior to initiating the disclosed therapies,for example to determine whether the subject has a tumor. The presenceof a tumor can be determined by methods known in the art, and typicallyinclude cytological and morphological evaluation. The tumor can be anestablished tumor. The cells can be in vivo or ex vivo, including cellsobtained from a biopsy. The presence of a tumor indicates that the tumorcan be treated using the methods provided herein. In some embodiments, asubject with a c-Myc-positive tumor is selected for treatment, forexample, by detecting c-Myc expression and/or activity in a biologicalsample obtained from the subject. For example, upregulated expression ofthe c-MYC gene (for example, as detected by an increase in c-Myc mRNA,c-Myc protein, or the expression of genes up-regulated by c-Myc comparedto a control) can be detected, and in some examples quantified. Thec-MYC gene expression in the biological sample is compared to a control(such as a normal, non-tumor sample). An increase in the expression ofthe c-MYC gene (such as an increase in c-Myc mRNA, c-Myc protein, or theexpression of genes up-regulated by c-Myc) in the biological samplerelative to the control indicates the presence of a c-Myc-positivetumor, and can be used to select a subject for treatment with one ormore of the compounds or compositions disclosed herein. For example, anincrease in the test sample of at least 50%, at least 75%, at least 80%,at least 90%, at least 100%, at least 200% or even greater than 500%,relative to the control, indicates the subject (such as a human subject)is likely to respond favorably to treatment with one or more of theagents disclosed herein. Suitable methods for detecting and/ormonitoring a c-Myc-positive tumor in a subject (such as a c-Myc-positivemultiple myeloma) can be selected by a treating physician. In oneembodiment, a sample is obtained from a subject, and the presence of acell that expresses c-Myc is assessed in vitro.

A therapeutically effective amount of a disclosed compound (such as anyof the compounds disclosed in section II, or one of Compounds 36-43) orcomposition containing same can be administered to a subject to treat atumor and/or cancer in the subject. The subject can be selected fortreatment that has, is suspected of having or is at risk of developing atumor or tumors, such as multiple myeloma or lymphoma. Subjects that canbenefit from the disclosed methods include, for example, human andveterinary subjects.

The administration of a compound (such as any of the compounds disclosedin section II, or one of Compounds 36-43) of the disclosure can be foreither prophylactic or therapeutic purpose. When providedprophylactically, the compound is provided in advance of any symptom.The prophylactic administration of the compound serves to prevent orameliorate any subsequent disease process. When providedtherapeutically, the compound is provided at (or shortly after) theonset of a symptom of disease or infection.

In some examples, a disclosed compound (such as any of the compoundsdisclosed in section II, or one of Compounds 36-43) or compositioncontaining same can be administered to a subject to slow or inhibit thegrowth or metastasis of a tumor and/or cancer. In these applications, atherapeutically effective amount of a disclosed compound (such as any ofthe compounds disclosed in section II, or one of Compounds 36-43) orcomposition containing same can be administered to a subject in anamount and under conditions sufficient to bind to the G4 present in thec-MYC promoter and reduce c-Myc expression, thereby slowing orinhibiting the growth or the metastasis of a tumor, or to inhibit a signor a symptom of a tumor. Examples of suitable subjects include thosediagnosed with or suspecting of having cancer (for example, a subjecthaving a tumor), for example a subject having a multiple myeloma.

The therapeutically effective amount will depend upon the severity ofthe disease and the general state of the patient's health. Atherapeutically effective amount is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inone embodiment, a therapeutically effective amount is the amountnecessary to inhibit tumor growth, or the amount that is effective atreducing a sign or a symptom of the tumor. The therapeutically effectiveamount of the agents administered can vary depending upon the desiredeffects and the subject to be treated. In some examples, therapeuticamounts are amounts which eliminate or reduce the patient's tumorburden, or which prevent or reduce the proliferation of metastaticcells.

The actual dosage of the compound will vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the compound for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of a compound and/or other biologically active agent within themethods and formulations of the disclosure is about 0.01 mg/kg bodyweight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, intraosseous, or intranasal delivery versus intravenous orsubcutaneous or intramuscular delivery. Dosage can also be adjustedbased on the release rate of the administered formulation, for example,of an intrapulmonary spray versus powder, sustained release oral versusinjected particulate or transdermal delivery formulations, and so forth.

Any method of administration can be used for the disclosed therapeuticagents, including local and systemic administration. For exampletopical, oral, intravascular such as intravenous, intramuscular,intraperitoneal, intranasal, intradermal, intrathecal and subcutaneousadministration can be used. The particular mode of administration andthe dosage regimen will be selected by the attending clinician, takinginto account the particulars of the case (for example the subject, thedisease, the disease state involved, and whether the treatment isprophylactic). In cases in which more than one agent or composition isbeing administered, one or more routes of administration may be used.

For prophylactic and therapeutic purposes, the compound can beadministered to the subject by the oral route or in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the compound can be provided as repeated doses within aprolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, avian, dog, sheep, porcine, feline, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using in vitro models. Using suchmodels, only ordinary calculations and adjustments are required todetermine an appropriate concentration and dose to administer atherapeutically effective amount of the compound (for example, amountsthat are effective to alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the compound may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

In some embodiments, local administration of the disclosed compounds canbe used, for instance by applying a disclosed compound to a region oftissue from which a tumor has been removed, or a region suspected ofbeing prone to tumor development. In some embodiments, sustainedintra-tumoral (or near-tumoral) release of the pharmaceuticalpreparation that includes a therapeutically effective amount of adisclosed compound may be beneficial.

The disclosed therapeutic agents can be formulated in unit dosage formsuitable for individual administration of precise dosages. In addition,the disclosed therapeutic agents may be administered in a single dose orin a multiple dose schedule. A multiple dose schedule is one in which aprimary course of treatment may be with more than one separate dose, forinstance 1-10 doses, followed by other doses given at subsequent timeintervals as needed to maintain or reinforce the action of thecompositions. Treatment can involve daily or multi-daily doses ofcompound(s) over a period of a few days to months, or even years. Thus,the dosage regime will also, at least in part, be determined based onthe particular needs of the subject to be treated and will be dependentupon the judgment of the administering practitioner.

In particular examples, the subject is administered a therapeuticcomposition that includes one or more of the disclosed compounds on amultiple daily dosing schedule, such as at least two consecutive days,10 consecutive days, and so forth, for example for a period of weeks,months, or years. In one example, the subject is administered theconjugates, antibodies, compositions or additional agents for a periodof at least 30 days, such as at least 2 months, at least 4 months, atleast 6 months, at least 12 months, at least 24 months, or at least 36months.

In some embodiments, the disclosed methods include providing surgery,radiation therapy, and/or chemotherapeutics to the subject incombination with administration of a disclosed compound or compositioncontaining same. Methods and therapeutic dosages of such agents andtreatments are known to those skilled in the art, and can be determinedby a skilled clinician. Preparation and dosing schedules for theadditional agent may be used according to manufacturer's instructions oras determined empirically by the skilled practitioner. Preparation anddosing schedules for such chemotherapy are also described inChemotherapy Service, (1992) Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md.

Non-limiting examples of additional therapeutic agents that can be usedwith the combination therapy include microtubule binding agents, DNAintercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNAtranscription inhibitors, antibodies, enzymes, enzyme inhibitors, generegulators, angiogenesis inhibitors, and proteosome inhibitors (such asbortezomib or carfilzomib). These agents (which are administered at atherapeutically effective amount) and treatments can be used alone or incombination. For example, any suitable anti-cancer or anti-angiogenicagent can be administered in combination with the antibodies, conjugatesdisclosed herein. Methods and therapeutic dosages of such agents areknown to those skilled in the art, and can be determined by a skilledclinician.

Additional chemotherapeutic agents include, but are not limited toalkylating agents, such as nitrogen mustards (for example, chlorambucil,chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas(for example, carmustine, fotemustine, lomustine, and streptozocin),platinum compounds (for example, carboplatin, cisplatin, oxaliplatin,and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine,temozolomide, thiotepa, and uramustine; antimetabolites, such as folicacid (for example, methotrexate, pemetrexed, and raltitrexed), purine(for example, cladribine, clofarabine, fludarabine, mercaptopurine, andtioguanine), pyrimidine (for example, capecitabine), cytarabine,fluorouracil, and gemcitabine; plant alkaloids, such as odophyllum (forexample, etoposide, and teniposide), taxane (for example, docetaxel andpaclitaxel), vinca (for example, vinblastine, vincristine, vindesine,and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracyclinefamily members (for example, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin,hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecanand irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab,cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, andtrastuzumab; photosensitizers, such as aminolevulinic acid, methylaminolevulinate, porfimer sodium, and verteporfin; and other agents,such as alitretinoin, altretamine, amsacrine, anagrelide, arsenictrioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib,celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib,hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin,masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib,vemurafinib, vandetanib, and tretinoin. Selection and therapeuticdosages of such agents are known to those skilled in the art, and can bedetermined by a skilled clinician.

The combination therapy may provide synergy and prove synergistic, thatis, the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation, a synergistic effect maybe attained when the compounds are administered or deliveredsequentially, for example by different injections in separate syringes.In general, during alternation, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

IV. Compositions

Another aspect of the disclosure includes pharmaceutical compositionsprepared for administration to a subject and which include atherapeutically effective amount of one or more of the compoundsdisclosed herein. The therapeutically effective amount of a disclosedcompound will depend on the route of administration, the species ofsubject and the physical characteristics of the subject being treated.Specific factors that can be taken into account include disease severityand stage, weight, diet and concurrent medications. The relationship ofthese factors to determining a therapeutically effective amount of thedisclosed compounds is understood by those of skill in the art.

Pharmaceutical compositions for administration to a subject can includeat least one further pharmaceutically acceptable additive such ascarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutically acceptablecarriers useful for these formulations are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 19th Edition (1995), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical compositions disclosed herein include those formed frompharmaceutically acceptable salts and/or solvates of the disclosedcompounds. Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Particular disclosed compounds possess at least one basic group that canform acid-base salts with acids. Examples of basic groups include, butare not limited to, amino and imino groups. Examples of inorganic acidsthat can form salts with such basic groups include, but are not limitedto, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuricacid or phosphoric acid. Basic groups also can form salts with organiccarboxylic acids, sulfonic acids, sulfo acids or phospho acids orN-substituted sulfamic acid, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconicacid, glucaric acid, glucuronic acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid or isonicotinic acid, and, in addition, with amino acids, forexample with α-amino acids, and also with methanesulfonic acid,ethanesulfonic acid, 2-hydroxymethanesulfonic acid,ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In particular, suitable salts includethose derived from alkali metals such as potassium and sodium, alkalineearth metals such as calcium and magnesium, among numerous other acidswell known in the pharmaceutical art.

Certain compounds include at least one acidic group that can form anacid-base salt with an inorganic or organic base. Examples of saltsformed from inorganic bases include salts of the presently disclosedcompounds with alkali metals such as potassium and sodium, alkalineearth metals, including calcium and magnesium and the like. Similarly,salts of acidic compounds with an organic base, such as an amine (asused herein terms that refer to amines should be understood to includetheir conjugate acids unless the context clearly indicates that the freeamine is intended) are contemplated, including salts formed with basicamino acids, aliphatic amines, heterocyclic amines, aromatic amines,pyridines, guanidines and amidines. Of the aliphatic amines, the acyclicaliphatic amines, and cyclic and acyclic di- and tri-alkyl amines areparticularly suitable for use in the disclosed compounds. In addition,quaternary ammonium counterions also can be used.

Particular examples of suitable amine bases (and their correspondingammonium ions) for use in the present compounds include, withoutlimitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane,diazabicycloundecene, N-methyl-N-ethylamine, diethylamine,triethylamine, diisopropylethylamine, mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additionalexamples of “pharmacologically acceptable salts,” see Berge et al., J.Pharm. Sci. 66:1 (1977).

Compounds disclosed herein can be crystallized and can be provided in asingle crystalline form or as a combination of different crystalpolymorphs. As such, the compounds can be provided in one or morephysical form, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms. Such differentphysical forms of the compounds can be prepared using, for exampledifferent solvents or different mixtures of solvents forrecrystallization. Alternatively or additionally, different polymorphscan be prepared, for example, by performing recrystallizations atdifferent temperatures and/or by altering cooling rates duringrecrystallization. The presence of polymorphs can be determined by X-raycrystallography, or in some cases by another spectroscopic technique,such as solid phase NMR spectroscopy, IR spectroscopy, or bydifferential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by avariety of mucosal administration modes, including by oral, rectal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to other surfaces. Optionally, the compositions can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-arterial, intra-articular,intraperitoneal, intrathecal, intracerebroventricular, or parenteralroutes. In other alternative embodiments, the compound can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the compound can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the compound. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubilityenhancing agents (for example, cyclodextrins and derivatives thereof),stabilizers (for example, serum albumin), and reducing agents (forexample, glutathione) can be included. Adjuvants, such as aluminumhydroxide (for example, Amphogel, Wyeth Laboratories, Madison, N.J.),Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, can be included inthe compositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The compound can be combined with the base or vehicle according to avariety of methods, and release of the compound can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the compound is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the compound can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compound can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the compound can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the compoundand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of diseases and other conditions inmammalian subjects. Kits for diagnostic use are also provided. In oneembodiment, these kits include a container or formulation that containsone or more of the compounds described herein. In one example, thiscomponent is formulated in a pharmaceutical preparation for delivery toa subject. The compound is optionally contained in a bulk dispensingcontainer or unit or multi-unit dosage form. Optional dispensing meanscan be provided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

EXEMPLARY EMBODIMENTS

Clause 1. A method of decreasing c-Myc expression in a cell, comprisingcontacting the cell with an effective amount of a compound, or apharmaceutically acceptable salt or ester thereof, having a structureof:

wherein A, B, and C are each independently selected from a 4 to 7membered cycloaliphatic, optionally-substituted heterocycloaliphatic,optionally-substituted aryl, or optionally-substituted heteroaryl; eachR¹ is independently selected from optionally-substituted lower alkyl; ais 0 to 2; R² is selected from hydroxyl, halogen, lower haloalkyl, oroptionally-substituted lower alkoxy; X is optionally-substituted methyl,ethyl or propyl; each R³ is independently selected from halogen, lowerhaloalkyl, or optionally-substituted lower alkyl; b is 0 to 6; each Y isindependently selected from optionally-substituted lower alkyl,optionally-substituted amide, optionally-substituted sulfonamide, oroptionally-substituted phosphoramide; c is 0 to 3; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0 to 5; R⁶ is selected fromhalogen, hydroxyl, lower haloalkyl, or optionally-substituted loweralkyl; Z is selected from carbon, oxygen, nitrogen, or sulfur; andwherein if Z is carbon or nitrogen, R⁷ is selected from hydroxyl,halogen, lower haloalkyl, or optionally-substituted lower alkoxy.

Clause 2. The method of clause 1, wherein A is a 6-memberedoptionally-substituted aryl or optionally-substituted heteroaryl.

Clause 3. The method of clause 1 or clause 2, wherein X is methyl.

Clause 4. The method of any one of the prior clauses, wherein B isoptionally-substituted N-heterocyclic or N-heterocyclic.

Clause 5. The method of any one of the prior clauses, wherein B isselected from optionally-substituted pyrrolidine, optionally-substitutedimidizolidine, optionally-substituted pryazolidine,optionally-substituted pyrrole, optionally-substituted diazole,optionally-substituted triazole, optionally-substituted piperidine,optionally-substituted pyridine, optionally-substituted diazine,optionally substituted triazine, optionally-substituted piperazine,optionally-substituted azepane, or optionally-substituted azepine.

Clause 6. The method of any one of the prior clauses, wherein Y isamide.

Clause 7. The method of any one of the prior clauses, wherein C is6-membered optionally-substituted aryl or optionally substitutedheteroaryl.

Clause 8. The method of clause 1, wherein the compound has a structureof:

wherein A and C are each independently selected from a 5 or 6 memberedoptionally-substituted aryl, or optionally-substituted heteroaryl; eachR¹ is independently selected from optionally-substituted lower alkyl; ais 0 to 2; R² is selected from hydroxyl, halogen, lower haloalkyl, oroptionally-substituted lower alkoxy; R⁸ and R⁹ together with the linkingnitrogen atom form a 4 to 7 membered optionally-substitutedN-heterocycloaliphatic ring; each R³ is independently selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; b is 0to 6; R⁴ is selected from halogen, lower haloalkyl, oroptionally-substituted lower alkyl; each R⁵ is independently selectedfrom halogen, lower haloalkyl, or optionally-substituted lower alkyl; dis 0 to 5; R⁶ is selected from halogen, hydroxyl, lower haloalkyl, oroptionally-substituted lower alkyl; Z is selected from nitrogen oroxygen; and wherein if Z is carbon or nitrogen, R⁷ is selected fromhydroxyl, halogen, lower haloalkyl, or optionally-substituted loweralkoxy, or is not present.

Clause 9. The method of clause 8, wherein R⁸ and R⁹ together with thelinking nitrogen atom form optionally-substituted pyrrolidine,optionally-substituted imidizolidine, optionally-substitutedpryazolidine, optionally-substituted piperidine, optionally-substitutedpiperazine, optionally-substituted azepane.

Clause 10. The method of any one of the prior clauses, wherein a is 0.

Clause 11. The method of any one of the prior clauses, wherein R² ishydroxyl.

Clause 12. The method of any one of the prior clauses, wherein R³ methyland b is 1.

Clause 13. The method of any one of the prior clauses, wherein R⁴ islower haloalkyl.

Clause 14. The method of clause 13, wherein R⁴ is trifluoromethyl.

Clause 15. The method of any one of the prior clauses, wherein d is 0.

Clause 16. The method of any one of the prior clauses, wherein R⁶ ismethyl.

Clause 17. The method of any one of the prior clauses, wherein Z isnitrogen.

Clause 18. The method of clause 1, wherein the compound is one ofcompounds 1, 3, 6, 7, 12, 13, or 17-25.

Clause 19. The method of any one of the prior clauses, wherein the cellis in vitro.

Clause 20. The method of any one of clauses 1-18, wherein the cell is invivo.

Clause 21. The method of any one of the prior clauses, whereincontacting the cell with the effective amount of the compound or thepharmaceutically acceptable salt or ester thereof decreases c-Mycexpression in the cell by at least 50% compared to a control.

Clause 22. The method of any one of the prior clauses, whereindecreasing expression of c-Myc in the cell decreases growth and/orproliferation of the cell.

Clause 23. The method of any one of the prior clauses, wherein the cellis a cell with overexpression of the c-MYC gene.

Clause 24. The method of any one of the prior clauses, wherein thecompound selectively binds to a G4 quadruplex nucleic acid moleculecomprising the sequence set forth as SEQ ID NO: 2 with a K_(d) of nomore than 5 μM.

Clause 25. The method of any one of clauses 1-18 or 20-22, wherein thecell is a tumor cell in the subject, the method further comprisingtreating or preventing a tumor in the subject, comprising the step of:

administering to a subject in need thereof a therapeutically effectiveamount of the compound, or the pharmaceutically acceptable salt or esterthereof, to decrease c-Myc expression in the tumor cell, therebytreating or preventing the tumor in the subject.

Clause 26. The method of clause 23, wherein the tumor is a lymphoma ormultiple myeloma.

Clause 27. The method of clauses 25 or clause 26, wherein treating thetumor comprises decreasing tumor volume; decreasing the number or sizeof metastases; or lessening a symptom of the tumor.

Clause 28. The method of any of clauses 25-27, further comprisingadministering a therapeutically effective amount of an additionalanti-cancer agent to the subject, particularly wherein the additionalanti-cancer agent is a proteasome inhibitor, for example bortezomib orcarfilzomib.

Clause 29. A compound, or a pharmaceutically acceptable salt or esterthereof, having a structure of:

wherein A, B, and C are each independently selected from a 4 to 7membered cycloaliphatic, optionally-substituted heterocycloaliphatic,optionally-substituted aryl, or optionally-substituted heteroaryl; eachR¹ is independently selected from optionally-substituted lower alkyl; ais 0 to 2; R² is selected from hydroxyl, halogen, lower haloalkyl, oroptionally-substituted lower alkoxy; X is optionally-substituted methyl,ethyl or propyl; each R³ is independently selected from halogen, lowerhaloalkyl, or optionally-substituted lower alkyl; b is 0 to 6; each Y isindependently selected from optionally-substituted lower alkyl,optionally-substituted amide, optionally-substituted sulfonamide, oroptionally-substituted phosphoramide; c is 0 to 3; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0 to 5; R⁶ is selected fromhalogen, hydroxyl, lower haloalkyl, or optionally-substituted loweralkyl; Z is selected from carbon, oxygen, nitrogen, or sulfur; andwherein if Z is carbon or nitrogen, R⁷ is selected from halogen, lowerhaloalkyl, or optionally-substituted lower alkoxy or is not present; andwith the proviso selected from one or more of: Z is not oxygen; Y is notamide; X is not CH₂; c is not 1; R² is not hydroxyl; R⁶ is not methyl; Bis not a 5-, 6-, or 7-membered N-heterocycloalkyl ring; and the compounddoes not comprise the structure set forth as any one of structures 1-13,or 17-20.

Clause 30. The compound of clause 29, wherein A is a 6-memberedoptionally-substituted aryl or optionally-substituted heteroaryl.

Clause 31. The compound of clause 29 or clause 30, wherein X is methyl.

Clause 32. The compound of any one of clauses 29-31, wherein B isoptionally-substituted N-heterocyclic or N-heterocyclic.

Clause 33. The compound of any one of clauses 29-32, wherein B isselected from optionally-substituted pyrrolidine, optionally-substitutedimidizolidine, optionally-substituted pryazolidine,optionally-substituted pyrrole, optionally-substituted diazole,optionally-substituted triazole, optionally-substituted piperidine,optionally-substituted pyridine, optionally-substituted diazine,optionally substituted triazine, optionally-substituted piperazine,optionally-substituted azepane, or optionally-substituted azepine.

Clause 34. The compound of any one of clauses 29-33, wherein Y is amide.

Clause 35. The compound of any one of clauses 29-34, wherein C is6-membered optionally-substituted aryl or optionally substitutedheteroaryl.

36. The compound of clause 28, wherein the compound has a structure of:

wherein A and C are each independently selected from a 5 or 6 memberedoptionally-substituted aryl, or optionally-substituted heteroaryl; eachR¹ is independently selected from hydrogen or optionally-substitutedlower alkyl; a is 0 to 2; R² is selected from hydroxyl, halogen, lowerhaloalkyl, or optionally-substituted lower alkoxy; R⁸ and R⁹ togetherwith the linking nitrogen atom form a 4 to 7 memberedoptionally-substituted N-heterocycloaliphatic ring; each R³ isindependently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; b is 0 to 6; R⁴ is selected fromhalogen, lower haloalkyl, or optionally-substituted lower alkyl; each R⁵is independently selected from hydrogen, halogen, lower haloalkyl, oroptionally-substituted lower alkyl; d is 0 to 5; R⁶ is selected fromhydrogen, halogen, hydroxyl, lower haloalkyl, or optionally-substitutedlower alkyl; Z is selected from nitrogen or oxygen; and wherein if Z iscarbon or nitrogen, R⁷ is selected from hydroxyl, halogen, lowerhaloalkyl, or optionally-substituted lower alkoxy, or is not present.

Clause 37. The compound of clause 36, wherein R⁸ and R⁹ together withthe linking nitrogen atom form optionally-substituted pyrrolidine,optionally-substituted imidizolidine, optionally-substitutedpryazolidine, optionally-substituted piperidine, optionally-substitutedpiperazine, optionally-substituted azepane.

Clause 38. The compound of any one of clauses 29-37, wherein each of R¹is hydrogen.

Clause 39. The compound of any one of clauses 29-38, wherein R² ishydroxyl.

Clause 40. The compound of any one of clauses 29-39, wherein R³ ishydrogen or methyl.

Clause 41. The compound of any one of clauses 29-40, wherein R⁴ is lowerhaloalkyl.

Clause 42. The compound of clause 41, wherein R⁴ is trifluoromethyl.

Clause 43. The compound of any one of clauses 29-41, wherein each of R⁵is hydrogen.

Clause 44. The compound of any one of clauses 29-43, wherein R⁶ ismethyl.

Clause 45. The compound of any one of clauses 29-44, wherein Z isnitrogen.

Clause 46. The compound of clause 29, wherein the compound is one ofcompounds 21-25.

Clause 47. A pharmaceutical composition comprising a compound of any oneof clauses 29-46, and at least one pharmaceutically acceptable additive.

Clause 48. The pharmaceutical composition of clause 47, comprising aunit dosage form of a therapeutic amount of the compound.

Clause 49. The pharmaceutical composition of clause 47 or clause 48,further comprising an additional anticancer agent, particularly whereinthe anticancer agent is a proteasome inhibitor, for example bortezomibor carfilzomib.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Selective Inhibition of c-Myc Expression Via Small MoleculeStabilization of G-Quadruplex DNA

The transcription factor c-Myc plays a pivotal role in cancer initiationand progression, however small molecules that selectively suppress itsfunction or expression are limiting. One potential route topharmacological inhibition of c-Myc is preventing its expression throughsmall molecule mediated stabilization of the G-quadruplex (G4) presentin its promoter. Here, a small molecule that binds to quadruplex DNA,and inhibits c-Myc expression in cell models is reported. A smallmolecule microarray screen was used to identify compounds that bothdirectly and selectively bind to the c-MYC G4. Surface plasmon resonance(SPR) and thermal melt assays confirmed that one molecule identified inthis screen binds reversibly to the G4 with single digit micromolaraffinity. Furthermore, evaluation of this compound in biochemical andcell-based assays demonstrates that the compound effectively silencesc-Myc transcription and translation via a quadruplex-dependent mechanismof action. Consistent with other studies of c-Myc inhibition, thecompound induces G1 arrest and is selectively toxic to c-Myc-drivencancer cell lines that contain a quadruplex in the promoter, withminimal effects on peripheral blood mononucleocytes or a cell linelacking the G4. Gene expression analysis demonstrates that c-Myc and anumber of c-Myc target genes are downregulated upon treatment with thiscompound, while several other known quadruplex-driven genes are notaffected. This work confirms that selective stabilization of the c-MYCG4 is a viable strategy for attenuating c-Myc expression and function.

The oncogenic transcription factor c-Myc has a pleiotropic role in awide range of cell processes (Bretones, et al., Biochimica et biophysicaacta 2014) and is deregulated in some 70% of human cancers (Beroukhim,et al., Nature 2010, 463, 899). However, targeting the c-Myc proteindirectly has proven to be difficult due to a lack of well-definedpockets amenable to small molecule binding (Berg, et al., Proc Natl AcadSci USA 2002, 99, 3830; Yin, et al., Oncogene 2003, 22, 6151; Huang, etal., Experimental hematology 2006, 34, 1480; Wang, et al., Mol CancerTher 2007, 6, 2399) and alternative mechanisms to inhibit c-Myc functionare desirable (Dang, et al., Seminars in cancer biology 2006, 16, 253;Balasubramanian, et al., Nat Rev Drug Discov 2011, 10, 261). One suchalternative mechanism is through stabilization of the G-quadruplex (G4)present in the c-MYC promoter region (Balasubramanian, et al., Nat RevDrug Discov 2011, 10, 261). G4s are guanine-rich noncanonicalHoogsteen-bonded nucleotide structures found in many RNA and DNAsequences (FIG. 1A) (Huppert and Balasubramanian, Nucleic Acids Res2007, 35, 406; Gray, et al., Nat Chem Biol 2014, 10, 313). For example,expression of the proto-oncogene c-MYC is regulated by a 27 base pair(Pu27) sequence found in the nuclease hypersensitive element III (1)region (NHEIII₁) of the c-MYC gene known to form a G4 (Gonzalez andHurley, Annual review of pharmacology and toxicology 2010, 50, 111).Formation of the quadruplex in this sequence is believed to result in a“kink” in the DNA that prevents the polymerase from continuing along itsreading frame, ultimately resulting in downregulation of the associatedgene (FIG. 1B) (Weitzmann, et al., J Biol Chem 1996, 271, 20958). Theuse of small molecules to stabilize the G4 conformation and consequentlydecrease c-Myc expression is an attractive therapeutic goal in cancerswhere c-Myc is overexpressed (Siddiqui-Jain, et al., Proc Natl Acad SciUSA 2002, 99, 11593).

There are a number of reported ligands that effectively stabilizequadruplex DNA according to structural and biophysical measurements(Dash, et al., Chemistry 2011, 17, 4571), however few are validated incellular models (Castillo-Gonzalez, et at, Curr Pharm Des 2013, 19,2164; Wei, et al., International journal of biological macromolecules2013, 57, 1). Additionally, though some quadruplex ligands silence c-MYCexpression in cells, they may not be selective (Nasiri, et al., ChemCommun (Camb) 2014, 50, 1704) and activity cannot always be attributedto a c-MYC quadruplex-dependent mechanism of action (Boddupally, et al.,J Med Chem 2012, 55, 6076). The only G4-stabilizing drug that hasadvanced to clinical trials is Quarfloxin (CX-3552, CylenePharmaceuticals, Tetragene). Quarfloxin effects apoptosis and cell deathin cancer cells, and its mechanism of action is believed to involve theinhibition of ribosomal RNA biogenesis via disruption of the interactionbetween nucleolin and G4s in ribosomal DNA (Drygin, et al., Cancer Res2009, 69, 7653). Furthermore, many other reported G4 ligands are duplexDNA intercalators, exhibit promiscuous reactivity, or bind toquadruplexes with greater than a 1:1 binding stoichiometry(Balasubramanian, et al., Nat Rev Drug Discov 2011, 10, 261; Drygin, etal., Cancer Res 2009, 69, 7653; Dai, et al., J Am Chem Soc 2011, 133,17673). As an example, TMPyP4, a commonly used reagent in quadruplexbinding studies, is a cationic porphyrin that binds to quadruplex DNA inmultiple fashions (Freyer, et al., Biophys J 2007, 92, 2007), and alsohas significant off-target activity (Sibata, et al., Expert opinion onpharmacotherapy 2001, 2, 917; Grand, C. L.; et al., Mol Cancer Ther2002, 1, 565; Mikami-Terao, et al., Cancer Lett 2008, 261, 226). Asecond prominent example is pyridostatin, a compound designed to bindall G4s in the cell (Koirala, et al., Nat Chem 2011, 3, 782; Muller, etal., Org Biomol Chem 2012, 10, 6537. Thus, new classes of potent,selective quadruplex ligands that are active in tissue culture modelswould be of substantial utility as reagents to study c-Myc biology aswell as potential therapeutics.

The identification and characterization of a new class of small moleculec-MYC G4 ligands using small molecule microarrays (SMMs) is provided inthis example. A SMM screen of 20,000 compounds using a fluorescentlytagged G4 DNA oligonucleotide derived from the NHEIII₁ region in thec-MYC promoter was performed (Gonzalez and Hurley, Annual review ofpharmacology and toxicology 2010, 50, 111). One of the small moleculesidentified in this screen inhibits c-MYC transcription in vitro in aG4-dependent fashion. Direct and reversible binding to c-MYC G4-DNA wasvalidated through SPR and thermal melt assays. Furthermore, decreasedc-Myc transcription correlated with decreased viability across a panelof multiple myeloma cell lines. Additionally, the compound has minimaleffects on cell viability in a Burkitt's Lymphoma cell line harboring ac-MYC translocation that deletes the G4, and has only modest toxicity innormal peripheral blood mononucleocytes. Finally, gene expressionanalysis demonstrates that the compound reduces the expression of c-MYCand c-Myc target genes, and does not alter the expression of severalother genes known to be controlled by G4s, thus indicating considerableselectivity.

Results and Discussion

In order to identify compounds that selectively bind to c-MYC quadruplexDNA, a small molecule microarray (SMM) screening approach was used(FIGS. 1, 2) (Bradner, et al., Nat Protoc 2006, 1, 2344; Duffner, etal., Curr Opin Chem Biol 2007, 11, 74; Kawasumi, et al., J InvestDermatol 2005, 124, A39; Koehler, et al., J Am Chem Soc 2003, 125, 8420;Miao, et al., J Comb Chem 2007, 9, 245; Stanton, et al., Nat Chem Biol2009, 5, 154; Vegas, et al., Angew Chem Int Edit 2007, 46, 7960).Briefly, a library of 20,000 compounds was covalently immobilized onglass slides using isocyanate surface chemistry, as previously described(Sztuba-Solinska, et al., J Am Chem Soc 2014, 136, 8402). Next, aCy5-labeled c-MYC G4 oligonucleotide derived from the NHEIII₁ region ofthe promoter (Gonzalez and Hurley, Annual review of pharmacology andtoxicology 2010, 50, 111) was annealed and incubated with the printedlibrary to identify discrete binding interactions (FIG. 2A). Inparallel, several other Cy5-labeled oligonucleotide structures(including RNA hairpins (Sztuba-Solinska, et al., J Am Chem Soc 2014,136, 8402), the FOXO binding domain (Carter and Brunet, Curr Biol 2007,17, R113), and CAG repeat DNA (Michlewski and Krzyzosiak, J Mol Biol2004, 340, 665)) were screened in an analogous manner, which served ascontrols. For each compound in the library, a composite Z-score wascalculated, and the c-MYC G4-incubated data set was compared to abuffer-incubated control data set. Compounds were considered hits iftheir composite Z-score was greater than three (representing threestandard deviations from the mean of the screening library), and if nofluorescence was observed in the buffer-incubated sample. Next, thesehit compounds were eliminated if they were also found to bind to othernon-homologous oligonucleotides investigated by the same technique.

Using these criteria, 32 unique hits were identified as bindingselectively to the c-MYC quadruplex structure, for a final hit rate of0.16%. A panel of the twelve most promising hits was selected forfurther analysis on the basis of Z-score, qualitative inspection ofmicroarray results, and compound availability (FIG. 8). Each of thesecompounds was evaluated for its capacity to functionally inhibit c-MYConcogene transcription and translation, and to reduce cancer cellviability in multiple myeloma cell lines. On the basis of thesepreliminary studies, Compound 1, a benzofuran structurally unlike anyreported quadruplex-binding small molecules, was identified as apromising candidate and it was selected for further in-depthcharacterization (FIG. 2B).

To assess the ability of Compound 1 to bind to the c-MYC G4 in solution,a CD-based thermal melt experiment was employed. After annealing, themolecular ellipticity of the c-MYC G4 was measured by circulardichroism, where a maximum was observed at 262 nm and a minimum wasobserved at 244 nm, thus confirming proper folding of theoligonucleotide into a parallel-stranded quadruplex (Mathad, et al.,Nucleic Acids Res 2011, 39, 9023) (see FIG. 9). Next, molecularellipticity was monitored at 262 nm as a function of temperature inorder to measure the melting temperature (T_(m)). Finally, a samplecontaining equimolar concentrations of Compound 1 and the c-MYC G4oligonucleotide was evaluated in the same experiment. Molecules thatproductively bind to the G4-DNA stabilize the structure and thereforeincrease its T_(m) (Murat, et al., Chemical Society reviews 2011, 40,5293). In the presence of Compound 1, the T_(m) of the G4-DNA increasedby 2.1 (±0.5) ° C. (FIG. 2B). This result confirms that the compoundbinds to and stabilizes quadruplex DNA.

In order to quantitatively assess the binding affinity of Compound 1with the c-MYC G4, surface plasmon resonance (SPR) experiments (DeCrescenzo, et al., Cell Mol Bioeng 2008, 1, 204) were performed using abiotinylated c-MYC G4 oligonucleotide. The oligonucleotide wasimmobilized to a streptavidin-coated chip, and binding was measured as afunction of concentration. (FIG. 2). This experiment demonstrates thatCompound 1 binds to the c-MYC G4 DNA with an equilibrium dissociationconstant (K_(d)) of 4.5±1.4 μM (FIG. 2C). Furthermore, Compound 1interacts with quadruplex DNA through a reversible binding interaction,and rapid, complete dissociation can be observed in the sensogram (FIG.2C). Significantly, the data fit well to a 1:1 binding isotherm and thesaturation binding measurement of ˜20 response units suggests that thebinding stoichiometry is likely 1:1. When the same experiment wasperformed with Quarfloxin, no evidence of binding was observed, pointingto a unique mechanism for transcriptional inhibition of c-Myc withCompound 1.

Next, a modified version of the PCR-stop assay was used to investigatethe ability of Compound 1 to inhibit c-MYC DNA amplification in aG4-dependent fashion (Lemarteleur, et al., Biochem Biophys Res Commun2004, 323, 802; Ou, et al., J Med Chem 2007, 50, 1465). A linear c-MYCPu27 (mutant) sequence can be PCR-amplified using normal thermal cyclingconditions. However, a G4-containing Pu27 (wild type) sequence blockspolymerase activity and inhibits formation of the PCR product. In thepresence of a quadruplex-stabilizing ligand, PCR amplification isinhibited further. Indeed, Compound 1 demonstrated dose-dependentinhibition of PCR amplification for the wild type Pu27 sequence atconcentrations comparable to the K_(d) measured by SPR. In contrast,Compound 1 had no effect at concentrations up to 100 μM on theamplification of a mutant sequence incapable of G4 formation (FIG. 3A).These data point to a G4-dependent mechanism of inhibition by the leadcompound in vitro.

To confirm a G4 specific mechanism of action in cells, the CA46Burkitt's Lymphoma line was used in an exon-specific assay, aspreviously reported (Boddupally, et al., J Med Chem 2012, 55, 6076). Formost cell lines, 85-90% of c-Myc expression is controlled by the G4located prior to exons 1 and 2 in the promoter. Furthermore, expressionpredominantly occurs following exon 2, due to a thousand-fold increasein transcription from this allele (Brown, et al., J Biol Chem 2011, 286,41018). The CA46 Burkitt's Lymphoma cell line is an exception to thistrend due to the existence of a chromosome (8:14) (Pelicci, et al., ProcNatl Acad Sci USA 1986, 83, 2984) translocation between exons 1 and 2,leaving exon 1 under G4 control. As a result, c-Myc expression from exon2 is G4 independent (FIG. 3B). This renders the overall cell lineresistant to G4-mediated c-Myc inhibition at the RNA and protein level,and proliferation should be uninhibited by G4-stabilizing agents. Thus,in the presence of a G4 stabilizing agent, c-Myc transcription from exon1 should be downregulated, while transcription from exon 2 should belargely unaffected. Using FAM-tagged exon specific TaqMan geneexpression assays with qPCR for c-MYC, it was demonstrated that Compound1 results in sustained downregulation of transcription from exon 1,which contains a quadruplex, while transcription from exon 2, which doesnot contain a quadruplex, is unaffected at treatment times up to 48hours (FIG. 3C). This effect is further evidenced by the observationthat while Compound 1 silences c-MYC in several other multiple myelomacell lines, the CA46 Burkitt's Lymphoma cell line is resistant toinhibition of c-Myc protein translation by Compound 1 (FIG. 3C, videinfra).

Since two thirds of multiple myeloma cases involve deregulated c-MYCexpression (Kuehl and Bergsagel, Blood 2012, 120, 2351; Shou, et al.,Proc Natl Acad Sci USA 2000, 97, 228), we elected to evaluate the cellviability effects of Compound 1 in a multiple myeloma model. Compound 1inhibited myeloma cell viability in a dose- and time-dependent manner,with an IC₅₀ of 7.6±1.1 μM after 72 hours (FIG. 4A). Furthermore,Compound 1 induced a seven-fold decrease in c-Myc transcription after 24hours (FIG. 4B). Additionally, c-Myc protein translation was alsosignificantly inhibited by exposure to 10 μM of Compound 1. Thissuppression was maintained over 72 hours, which is notable given thecharacteristic rapid replenishment of the protein—a phenomenon thatcomplicates targeting c-Myc at the protein level (FIG. 4C). This effectwas maintained across a genetically diverse set of 4 multiple myelomalines. We observed decreases in c-MYC translation in all lines tested,and this effect also correlated with decreases in viability (FIG. 4D).The CA46 Burkitt's Lymphoma line (lacking a G4) was included in thispanel as a resistant control, as it showed negligible changes in c-MYCexpression or cell viability when treated with Compound 1. Notably,Compound 1 did not alter viability in peripheral blood mononucleocytesdrawn from a healthy volunteer, even at doses 25% higher than those usedfor the cell treatments discussed above (FIG. 4E).

To further explore the mechanism of action for the anticancer activityof Compound 1, cell cycle analysis was performed and monitored forpotential induction of apoptotic death. Compound 1 was observed tocauses ˜85% of treated cells to arrest in the G1 phase after 72 hours,as measured by propidium iodide staining (FIG. 5A). Additionally,minimal increases in apoptosis were observed, by Annexin V/7-AADstaining and FACS analysis; only 13% of the cells were undergoingapoptosis (FIG. 5B). These results may be indicative of the lownonspecific cytotoxicity and high target specificity of Compound 1,which is important in achieving a useful therapeutic window.Additionally, Compound 1 triggered a senescent state in a majority oftreated myeloma cells after 72 hours, supporting cell cycle arrest as aprimary mechanism of action (FIG. 5C). Taken together, these resultssupport that Compound 1 is acting through suppression of c-MYCexpression, rather than a non-specific mechanism of action.

Having demonstrated that Compound 1 suppresses c-MYC expression,subsequent experiments focused on gaining insight into the specificityof Compound 1 by probing effects on gene expression in a broader sense.Gene expression analysis was performed on a panel of 770cancer-associated genes in a multiplexed (nanostring) transcriptionalassay. In addition to c-MYC itself, several c-Myc target genes wereincluded in the panel, as were a number of other quadruplex-drivengenes. Cells were treated with 10 μM Compound 1 for 2h, 4h, 12h, 24h,and 48h, and separately with doses of 1, 2.5, 5, and 10 μM at a timepoint of 24h, and the effects on gene expression were evaluated. c-MYCwas one of the most suppressed genes, and a number of known c-Myc targetgenes were also suppressed, including E2F1, MCM2, MCM4, MCM5, andCDCl25A. Additionally, a third data set was collected comparing Compound1 treatment to JQ-1 (a BET-bromodomain inhibitor) and quarfloxin(another quadruplex-binding small molecule). All three inhibitorsexhibited substantial differences in gene expression profiles,highlighting a unique mechanism of action of Compound 1.

The effects of Compound 1 on a number of known quadruplex-driven genesin the panel were examined further (FIG. 6). Expression levels of MYC,RB1, VEGFA, KRAS, and HIF1α, all of which are reported to be under thecontrol of promoter G4s, and are expressed in L363 cells, were includedin the panel of genes evaluated by Nanostring as discussed above. Thechange in expression for each of these genes (nanostring) over time ispresented in FIG. 6A. While MYC expression was substantially reduced atall time points, expression of other G4-associated genes was minimallyaffected. To further confirm these results, we also performed qPCRexperiments on these genes following treatment with Compound 1 (FIG.6B). Again, MYC expression is greatly reduced while other genes areminimally affected. These changes in gene expression are in line withbiophysical measurements of compound affinity. Corresponding datashowing similar results is presented for Compounds 1 and 23 in FIGS. 14Aand 14B. While the quadruplex with the highest affinity for Compound 1(MYC) had pronounced changes in gene expression, G4s with weaker binding(RB1, BCL2) or a complete lack of G4 binding (KRAS, VEGF) had minimalchanges in expression, even after 48 h of treatment. These observationsfurther highlight the ability of Compound 1 and Compound 23 tospecifically target the expression of c-Myc and related pathways.

Having established the reversible binding of Compound 1 to quadruplexDNA and selective quadruplex-dependent silencing of c-Myc, oneconsideration that remained was the presence of a benzylaminophenolfunctional group in Compound 1. It has previously been reported thatcompounds containing this functional group can have the propensity toeject amines, form an o-quinone methide, and alkylate proteins (Weinert,et al., J Am Chem Soc 2006, 128, 11940; Herzig, et al., J Org Chem 2006,71, 4130; McLean, et al., Bioorg Med Chem Lett 2009, 19, 6717). Toassess whether Compound 1 was undergoing this reactivity, a small seriesof analogs was prepared. In FIG. 6C, compounds lacking the phenol oramino group were prepared and evaluated. As can be observed, a compoundincapable of forming a quinone methide retains activity in silencingc-MYC expression, while a compound lacking the amino substitution is notactive, demonstrating the importance of the amino group for activity.Additionally, the stability of Compound 1 was evaluated (see FIG. 7).Here, Compound 1 persisted in culture media over a period of 72 h asmonitored by LC/MS, confirming that the compound is largely stable incomplex, biologically relevant mixtures over the timeframe of viabilityassays used in this study. Additionally, the putative hydrolysisproducts arising from quinone methide formation were not observed at anytime by LC/MS. Taken together, these data point toward a reversiblebinding interaction not dependent on the formation of a quinone methide.

CONCLUSION

The overexpression of c-MYC is implicated in a vast number of humancancers, however only a small number of inhibitors have been describedin the literature, and as of now none are clinically approved.Pharmacological inhibition of c-Myc has historically been a challenge,and small molecules that are efficacious in cells are relatively rare.G4-DNA stabilization and subsequent transcriptional silencing by a smallmolecule is an attractive strategy for c-MYC inhibition because itcircumvents targeting c-Myc at the protein level. Nevertheless, many ofthe compounds currently known to bind to the c-MYC G4 generally havepoor drug-like properties, nonspecific quadruplex binding, or exhibitquadruplex-independent effects in more complex cell culture models.

To address challenges in the identification of c-MYC G4-bindingcompounds, a small molecule microarray-based screening approach wasemployed. By evaluating multiple oligonucleotide structuressimultaneously as part of the initial screen, selectivity considerationswere incorporated early in the discovery process. Through evaluation ofthe most promising hits in preliminary assays, Compound 1 was pursuedfor further investigation. The benzofuran structure of Compound 1 is anovel quadruplex-binding small molecule scaffold. Molecules containingbenzylaminophenol groups have previously been reported to form quinonemethide structures in aqueous solution. However, in this case astability study demonstrated that the compound persists for three daysin culture media. Furthermore, reversible binding was demonstrated bySPR experiments, where a K_(d) of 4.5±1.4 μM was measured, with anapparent 1:1 binding stoichiometry. By evaluating a small panel ofanalogs of Compound 1, further information about the pharmacophore wasgained. A compound lacking the amine was completely inactive, while ananalog lacking the phenol group (incapable of forming a quinone methide)retained activity. Thus, the amine functionality is likely required forbinding to the quadruplex, while the formation of a quinone methidestructure is unlikely to be involved in the mechanism. In contrast toquinone methide-forming alkylators, which are typically non-specificallytoxic, Compound 1 displays no toxicity to the CA46 resistant cell lineor peripheral blood mononucleocytes at relevant concentrations, furthersuggesting on-target activity as the origin of the observed effects incells.

In culture models, Compound 1 inhibits c-MYC expression at both thetranscriptional and translational levels. Moreover, it decreased theviability of several myeloma cell lines in a dose dependent fashion.Cell cycle analysis demonstrates that Compound 1 also triggers G1 arrestand senescence in myeloma cells, which is consistent with literaturefindings regarding effects of c-MYC knockdown on cell cycle progression(Wang, et al., Oncogene 2008, 27, 1905). In sum, these results validatethat Compound 1 effectively suppresses c-MYC expression through aG4-dependent inhibitory mechanism in vitro and in cancer cells.

Through gene expression profiling, further evidence for the suppressionof c-Myc and related proteins was observed. In a panel of 770cancer-related genes, the expression of c-MYC itself and a number ofc-Myc target genes were suppressed in a dose- and time-dependent manner.In comparison to quarfloxin and JQ1, Compound 1 appears to have a uniquemechanism of action and modulates a distinct subset of genes.Importantly, in depth analysis of the data set indicated that severalother known quadruplex-driven genes were not affected by Compound 1.Thus, Compound 1 has specific effects for the c-MYC quadruplex overseveral other quadruplex sequences within the genome. Furthermore,transcriptional profiling provides evidence for the selective modulationof the c-MYC pathway in a broader sense.

Materials and Methods

General Materials and Methods. Reactions were conducted using anhydroussolvents (passed through activated alumina columns). All commerciallyobtained reagents were used as received. Flash column chromatography wasperformed using normal phase silica gel (60 Å, 230-400 mesh, RediSep®Normal-phase Silica Flash Columns) on a CombiFlash® Rf 200i (TeledyneIsco Inc). Preparative HPLC was performed with a Waters® 2545 BinaryGradient Module equipped with a Waters® 2767 Sample Manager fractioncollector and a Luna 10 μm C18 110 Å (75×30 mm) column obtained fromPhenomenex, Inc. High-resolution LC/MS analyses were conducted on aThermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer system with anIon MAX API electrospray ion source in positive ion mode. AnalyticalLC/MS was performed using a Shimadzu LCMS-2020 Single Quadrupoleutilizing a Kinetex 2.6 μm C18 100 Å (2.1×50 mm) column obtained fromPhenomenex Inc. Runs employed a gradient of 0→90% MeOH/0.1% aqueousformic acid over 4.5 min at a flow rate of 0.2 mL/min. ¹H NMR and ¹³CNMR spectra were recorded on Varian and Bruker spectrometers (at 400 or500 MHz or at 100 or 125 MHz) and are reported relative to deuteratedsolvent signals. Data for ¹³H NMR spectra are reported as follows:chemical shift (δ ppm), multiplicity, coupling constant (Hz), andintegration. Data for ¹³C NMR spectra are reported in terms of chemicalshift. Surface plasmon resonance analysis was performed at the ATRF(NCI-Frederick) using a Biacore T200 (GE Healthcare).

Small Molecule Microarray Screening. Small molecule microarray screeningwas carried out as previously described (Duffner, et al., Curr Opin ChemBiol 2007, 11, 74; Sztuba-Solinska, et al., J Am Chem Soc 2014, 136,8402; Bradner, et al., Nat Protoc 2006, 1, 2344). Briefly, γ-aminopropylsilane (GAPS) microscope slides were functionalized with a shortFmoc-protected amino polyethylene glycol spacer. After deprotectionusing piperidine, 1,6-diisocyanatohexane was coupled to the surface byurea bond formation to provide functionalized isocyanate-coatedmicroarray slides that can react with primary alcohols and amines toform immobilized chemical screening libraries. 20,000 unique smallmolecule stock solutions (10 mM in DMSO) purchased from ChemBridge andChemDiv screening libraries, in addition to dyes and controls, wereprinted in duplicate onto four slides of 5,000 compounds each, andexposed to pyridine vapor to facilitate covalent attachment to the slidesurface. After drying, slides were incubated with a polyethylene glycolsolution to quench unreacted isocyanate surface. Printed slides wereincubated for 1 h at room temperature with a Cy5-tagged DNAoligonucleotide of the c-MYC G-quadruplex forming sequence(5′d(Cy5)-TGAGGGTGGGTAGGGTGGGTAA-3′, SEQ ID NO: 2), which had beenannealed by heating to 95° C. for three minutes, cooled to roomtemperature, and diluted to 500 nM in PBS. Following incubation, slideswere gently washed three times for 5 min in PBST, twice in PBS, and oncein deionized water to remove unbound oligonucleotide, and dried bycentrifugation for 2 min at 3400 g. Fluorescence intensity was measured(650 nm excitation, 670 nm emission) on a GenePix 4000a MicroarrayScanner. Hits were identified on the basis of signal-to-noise ratio(SNR), defined as (mean foreground−mean background)/(standard deviationof background), and Z-score, with the following criteria: (1) Raw SNR>0,(2) SNR>3 SD above negative control readings, (3) coefficient ofvariance (CV) of replicate spots<100, (4) SNR of negative controlslide<1, and (5) no activity with any other nucleic acid structuresscreened. The other nucleic acids were the FOXO3 DNA transcriptionfactor binding domain, CAG DNA repeat, HIV TAR RNA, and miR-21 RNA, allof which were Cy5-labeled, and the screens were run in the same methoddescribed above using the respective Cy5-nucleic acid instead of thec-MYC DNA.

PCR Stop Assay. A test oligonucleotide and a complementary sequence thatpartially hybridizes to its last G-repeat (sequences below) weresynthesized by IDT. The reactions were performed in a master mixcontaining 1× PCR buffer, 10 μmol of each oligo, 0.16 mM dNTP, 1.5 mMMgCl₂, 2.5 U HotStarTaq polymerase (Qiagen), and a dose titration of aligand of interest, spanning three orders of magnitude, in 25 μL totalvolume. The thermal cycling conditions were as follows: 94° C. for 5min, followed by 22 cycles of 94° C. for 30 s, 58° C. for 30 s, 72° C.for 30 s, and finally held at 4° C. following completion. The amplifiedproducts were mixed with 6×DNA Loading Dye (Thermo Scientific) andresolved on a 15% TBE-Urea Gel (Invitrogen) on the Novex mini gel systemat 150 V for 1 h. The gel products were stained in a 0.01% (v/v).Ethidium Bromide-TBE solution for 15 min and imaged under UV light onthe GBOX F3 (Syngene).

Oligos Used:

Forward 5′-AGG GTG GGG AGG GTG GGG-3′, nucleotides 3-19 of SEQ ID NO: 2(Partial sequence in the promoter of oncogene c-MYC that may formG-quadruplex.) Forward Mutant 5′-AGG GTG AAA AGG GTG GGG-3′, SEQ ID NO:3

Reverse 5′-ATC GAT CGC TTC TCG TCC TTC CCC A-3′, SEQ ID NO: 4(Complementary sequence used for both forward and reverse.)

Exon Specific Assay. CA46 cells were treated with ligands of interest orDMSO control at designated time points, washed in PBS, flash frozen, andRNA isolated using the Qiagen RNeasy Kit. RNA was quantified byNanoDrop, and 0.5 μg was reverse transcribed for use in qPCR. Reversetranscription was performed using the Applied Biosystems Kit B808-0234,cycled at 25° C. for 10 min, 48° C. for 60 min, 95° C. for 5 min, andheld at 4° C. following completion in 25 μL total volumes. The cDNA wasdiluted four fold and used in qPCR with the Taqman Gene ExpressionAssays (Life Technologies, exon 1: 01562521_m1, exon 2: 00153408_m1),cycled at 50° C. for 2 min, 95° C. for 10 min, and followed by 40 cyclesof 95° C. for 15 s and 60° C. for 1 min on the Applied Biosystems 7500Fast Real-Time PCR System. For exon 1 and exon 2, ΔC_(t) was normalizedto a VIC-Primer Limited tagged GAPDH Taqman Gene Expression Assay(multiplexed in the same well) and DMSO treated control samples.

CD-Thermal Melt Assay. Thermal stability of the c-MYC G4-formingoligonucleotide Pu22 (TGAGGGTGGGTAGGGTGGGTAA, SEQ ID NO: 2) in theabsence and presence of compounds was recorded on an Aviv BiomedicalInc. Model 420 Circular Dichroism Spectrometer. The c-MYC G-quadruplexwas diluted to 50 μM in 10 mM Tris buffer (pH 7.5, containing 100 mMKCl), heated to 95° C. for 5 min, and allowed to cool to roomtemperature. Positive molecular ellipticity of the parallel G-quadruplexpeak (262 nm) was confirmed by spectral examination. To 150 μL of theG-quadruplex in buffer was added one equiv. of compound (150 μL of a 50μM solution in buffer containing 0.5% DMSO), after which the mixtureswere heated from 5 to 95° C. at 2° C./min in a 0.1 mm quartz cell.Molecular ellipticity as a function of temperature was used to calculatea T., (the temperature at which 50% of the formed higher order DNAstructure was melted) for each condition using GraphPad Prism 6 softwareand a nonlinear regression model with a variable slope. ΔT_(m) valueswere calculated as T_(m(+compound))−T_(m(control)).

Surface Plasmon Resonance (SPR). SPR experiments were performed with aBiacore T200 (GE Healthcare). 20 μg/mL biotin-labeled Pu22 G-quadruplexin 10 mM Tris buffer (pH 7.5, containing 100 mM KCl, 3 mM EDTA) washeated to 95° C. for 5 min, and allowed to cool to room temperature. Thec-MYC DNA was then captured (FC2: 1245 Ru) on a Series S Sensor CM5 Chip(GE Healthcare) with amine-coupled Neutravidin (FC1: 4466 Ru, FC2: 5458Ru). Single Cycle Kinetics (SCK) experiments were carried out with fiveinjections of increasing concentration of analyte solution, which wasprepared by a three-fold serial dilution of compound with buffer (10 mMTris pH 7.5, 100 mM KCl, 3 mM EDTA) containing 3% DMSO. Binding analysiswas conducted at a flow rate of 30 μL/min at 25° C. In each run, theassociation phase and the subsequent dissociation phase were monitoredfor 1 min and 10 min, respectively. Prior to each compound injection,three buffer injections were made. From the obtainedreference-subtracted sensorgrams, the dissociation constants (K_(d)) ofthe compounds were estimated by a global fitting to a simple 1:1 bindingmodel in the Biacore evaluation software (GE Healthcare).

Cell Culture Conditions and Experimental Endpoints. Human multiplemyeloma and Burkitt's Lymphoma cell lines L363, CA46, MM-1R, KMS-11, andMM-1S were cultured and authenticated as previously described (Simmons,et al., Molecular oncology 2014, 8, 261). All plasma derived cell lineswere cultured in RPMI-1640 (2 mM L-glutamine, 10% fetal bovine serum(FBS), 100 U/ml penicillin, 100 μg/ml streptomycin: Gibco) and incubatedat 37° C. with 5% CO₂. Viability experiments were performed inquadruplicate on 96-well plates (Costar) at designated time and dosepoints. MTS reagent was then directly added, incubated at 37° C. for 90min, and absorbance of MTS formazan was read at 500 nm on an Omega 640spectrophotometer. Percentage cell viability was normalized to theabsorbance of untreated (DMSO) wells. In the case of cells harvested fortheir protein or RNA, pellets were flash frozen and stored at −80° C.overnight prior to use.

Western Blots. Cell pellets/tissue were homogenized and lysed in RIPAbuffer on ice for 1 h. Protein was quantitated by BCA, and equal proteinwas loaded onto 4-12% Bis-Tris Gels (Novex), electrophoresed at 150 Vfor 75 min to obtain sufficient separation, and transferred via theiBlot system (Life Technologies). Successful transfer and uniformloading was confirmed by Ponceau S staining (Thermo Scientific). Blotswere blocked in 10% dry milk in TBST, incubated with primary monoclonalantibodies in 5% BSA at concentrations designated by the manufacturer,and gently rotated at 4° C. overnight. Blots were washed with TBST threetimes prior to incubation with polyclonal secondary antibodies for 1 hin 5% dry milk at room temperature. Blots were washed three more timeswith TBST and imaged with Supersignal West Dura ChemiluminescentSubstrate (Thermo Scientific) on the GBOX F3 (Syngene). The c-Mycmonoclonal antibody was purchased from abcam (ab84132) and used at aconcentration of 1:1000. All other monoclonal antibodies were purchasedfrom Cell Signaling Technologies and used at a concentration of 1:1000,with the exception of α-β tubulin, which was used at a concentration of1:2000. All primary antibodies used in this study were of rabbit origin,and goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate(Invitrogen G21234) was used as the polyclonal secondary antibody at aconcentration of 1:4000.

Cancer Genome-Wide Probing and Statistical Packaging. The phenotype forc-MYC genetic knockdown in multiple myeloma was achieved throughlentiviral transduction of shRNA for c-MYC, derived from thespinoculation of 293T cells and plasmids grown in STBL3 E. coli, andisolated with the Qiagen Plasmid Midi Kit. Aliquots of the plasmids weregenerously provided by Art Shaffer. RNA of treated myeloma cells atdesignated time points was isolated with the Qiagen RNeasy kit, and usedwith the nCounter Human Cancer Reference Kit (NanoString Technologies),surveying changes in expression for 780 cancer-related human genes and 6reference genes. Quantitative changes in expression were analyzed andgrouped in the form of a heat map using the programming language, R. Allother quantitative statistical packing was performed in GraphPad Prism.

Stability Study. Compound 1 (10 μL of 1 mM DMSO solution) was added to490 μL RPMI-1640 culture media. After 1 min, 25 h, 48 h, and 72 h, 100μL of this solution was diluted into 100 μL acetonitrile. The mixturewas centrifuged at 5500 rpm for 1 min and the supernatant was removedfrom the pellet. Another 800 μL MeCN was added to the supernatant andthe mixture was centrifuged again at 5500 rpm for 1 min. The resultingsupernatant was subjected to LC/MS (ESI⁺) on an Agilent Technologies1200 LC/MSD single quadrupole system, equipped with an in-linediode-array UV detector. The mass corresponding to Compound 1 (M+H⁺=393)was extracted. This mass persisted as a significant peak through 72 h.Masses of the putative hydrolyzed compound (see structure below;M+H⁺=312) and o-quinone methide compound (see structure below; M+H⁺=294)were extracted, and were found to be insignificant at all time points(see the chromatograms below).

Synthesis of analogs. A general procedure for synthesis of certainanalogs of Compound 1 is shown below:

Additional details are provided below.

A solution of phenol (Otava 7018860558, 12 mg, 0.028 mmol) in DCM (0.28mL) was cooled to −78° C. Pyridine (4.5 μL, 0.056 mmol) was added andthe solution was stirred for 10 min. At this pointtrifluoromethanesulfonic anhydride solution (1M in DCM, 34 μL, 0.034mmol) was added slowly. The reaction was gradually warmed to rtovernight. After a total of 23 h, the solvent was removed in vacuo andthe residue was purified by flash column chromatography (10-50%EtOAc/hexanes) to provide X (9.5 mg, 65%) as a white solid. R_(f)=0.71(33% EtOAc/hexanes). ¹H NMR ((CD₃)₂SO, 400 MHz) δ 10.35 (s, 1H), 7.70(d, J=9.0 Hz, 1H), 7.67 (d, J=8.4 Hz, 2H), 7.32 (d, J=9.0 Hz, 1H), 7.16(d, J=8.3 Hz, 2H), 3.83 (s, 2H), 2.59 (s, 3H), 2.38-2.31 (m, 4H), 2.29(s, 3H), 1.41-1.34 (m, 4H), 1.34-1.25 (m, 4H). ¹³C NMR ((CD₃)₂SO, 125MHz) δ 161.0, 157.5, 151.8, 144.0, 136.9, 132.3, 129.1, 127.3, 125.8,119.1, 118.1 (q, J_(C-F)=320 Hz), 117.1, 116.5, 111.5, 54.4, 51.7, 26.8,26.2, 20.5, 13.5; HRMS (ESI) calculated for C₂₅H₂₈N₂O₆S (MH⁺) 541.1615,observed 541.1615.

To a solution of triflate X (3.1 mg, 0.0059 mmol) in DMF (60 μL, 0.098M) was added formic acid (0.56 μL, 0.014 mmol), tributylamine (5 μL,0.063 mmol), and Pd(PPh₃)₂Cl₂ (0.5 mg, 0.0007 mmol) successively underAr. The solution was heated to 110° C. for 1 h at which point it wascooled to room temperature. The mixture was filtered through cotton andthe filtrate was purified by HPLC (10-95% MeCN/H2O containing 0.1% TFAin the running buffer) to provide X as a white solid (2.5 mg, 86%). ¹HNMR ((CD₃)₂SO, 500 MHz) δ 10.64 (s, 1H), 9.48 (brs, 1H), 7.76 (t, J=4.5Hz, 1H), 7.63 (d, J=7.9 Hz, 2H), 7.45 (d, J=4.6 Hz, 2H), 7.23 (d, J=7.9Hz, 1H), 4.60 (d, J=5.3 Hz, 2H), 3.28-3.09 (m, 4H), 2.66 (s, 3H), 2.31(s, 3H), 1.89-1.69 (m, 4H), 1.67-1.47 (m, 4H); ¹³C NMR ((CD₃)₂SO, 125MHz) δ 165.1, 163.3, 157.5, 153.5, 135.8, 133.8, 129.3, 128.9, 128.2,124.8, 120.4, 119.1, 117.4 (q_(C-F), J=301.8 Hz), 113.8, 112.8, 54.5,53.7, 27.3, 26.1, 20.5, 14.0; HRMS (ESI) calculated for C₂₄H₂₉N₂O₂ (MH⁺)377.2224, observed 377.2225.

To a solution of 5-methoxy-2-methylbenzofuran-3-carboxylic acid (1.20 g,5.80 mmol) in DMF (29 mL) was added HATU (5.50 g, 14.0 mmol),p-toluidine (2.50 g, 23.0 mmol), and DIPEA (1.5 mL. 8.7 mmol). The brownsolution was heated to 80° C. under argon. After 22 h, the reaction wascooled to room temperature, after which it was diluted with EtOAc andwashed with NaHCO₃ (sat., aq). The aqueous layer was extracted 2× withEtOAc and the combined organic layers were washed successively withNH₄Cl (sat., aq.), brine, and H₂O (3×). It was then dried over Na₂SO₄,filtered and concentrated to give a brown oil. Flash columnchromatography (5-30% EtOAc/hexanes) afforded a fraction of pure X inaddition to a mixture of X and p-toluidine. The mixed fraction wasrepurified using the same conditions, and this process was repeated twoadditional times. In total 1.07 g (3.62 mmol, 62%) pure X was obtainedas a tan solid. R_(f)=0.60 (25% EtOAc/hexanes). ¹H NMR (CDCl3, 500 MHz)δ 7.63 (brs, 1H), 7.50 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.9 Hz, 1H), 7.17(d, J=8.2 Hz, 2H), 7.14 (d, J=2.5 Hz, 1H), 6.86 (dd, J=8.9, 2.5 Hz),3.82 (s, 3H), 2.68 (s, 3H), 2.35 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ162.4, 160.6, 156.6, 148.6, 135.3, 134.3, 129.7, 126.3, 120.4, 112.7,112.3, 111.8, 102.7, 56.0, 21.0, 14.2; HRMS (ESI) calculated forC₁₈H₁₈NO₃ (MH⁺) 296.1281, observed 296.1285.

A solution of X (703 mg, 2.38 mmol) in DCM (60 mL) was cooled to −78° C.Then BBr₃ (1M in DCM, 11.9 mL) was added slowly. The solution wasallowed to gradually warm to room temperature overnight. After a totalof 22 h˜60 mL 1N HCl added and the resulting mixture was stirredvigorously for 45 min. At this point, the DCM was removed in vacuo andthe aqueous mixture was extracted two times with EtOAc. The combinedorganic layers were washed with NaHCO₃ (sat., aq) followed by brine. Itwas then dried over Na₂SO₄, filtered, and concentrated to give a yellowsolid. Flash column chromatography (0-15% MeOH/DCM) afforded X (189 mg,28%) as a tan solid. R_(f)=0.32 (5% MeOH/DCM); ¹H NMR (CD₃OD, 500 MHz) δ7.53 (d, J=8.3 Hz, 2H), 7.27 (d, J=8.8 Hz, 1H), 7.17 (d, J=8.3 Hz, 2H),7.10 (d, J=2.4 Hz, 1H), 6.76 (dd, J=8.8, 2.5 Hz, 1H), 2.62 (s, 3H), 2.33(s, 3H); ¹³C NMR (CD₃OD, 125 MHz) δ 165.1, 160.0, 155.0, 149.5, 137.1,135.3, 130.3, 128.3, 122.0, 114.5, 113.9, 112.1, 106.1, 21.0, 13.9; HRMS(ESI) calculated for C₁₇H₁₆NO₃ (MH⁺) 282.1125, observed 282.1124.

Stability Study

Compound 1 (10 μL of 1 mM DMSO solution) was added to 490 μL RPMI-1640culture media. After 1 min, 25 h, 48 h, and 72 h, 100 μL of thissolution was diluted into 100 μL acetonitrile. The mixture wascentrifuged at 5500 rpm for 1 min and the supernatant was removed fromthe pellet. Another 800 μL MeCN was added to the supernatant and themixture was centrifuged again at 5500 rpm for 1 min. The resultingsupernatant was subjected to LC/MS (ESI⁺) on an Agilent Technologies1200 LC/MSD single quadrupole system, equipped with an in-linediode-array UV detector. The mass corresponding to Compound 1 (M+H⁺=393)was extracted and the chromatogram evaluated. This mass persisted as asignificant peak through 72 h. Masses of the putative hydrolyzedcompound (compound 26, see structure below; M+H⁺=312) and o-quinonemethide compound (see structure below; M+H⁺=294) were extracted, andwere found to be insignificant at all time points.

Example 2 Exemplary Analogs of Compound 1

FIG. 11 shows exemplary substitutions to Compound 1 that can be made togenerate analogs of Compound 1 that selectively bind to G4 quadruplexDNA comprising the sequence set forth as SEQ ID NO: 2, and can be usedto reduce c-MYC expression in cells. FIG. 10 illustrates an exemplaryprocedure for generating analogs of Compound 1. The amine group ofCompound 1 can be varied in reductive aminations to provide additionalanalogs. Several exemplary analogs of Compound 1 are listed in thefollowing table, which also shows results of assays for inhibition ofc-MYC expression in L363 cells (Peggy) and for viability of L363 cellsas discussed in Example 1. The table also provides an indication of anexemplary commercial source for the compounds, or (if applicable) anindication that the compound is new.

Peggy (% c-Myc expression Compound compared to No. StructureName/Catalog No. untreated) IC₅₀ 1

Commercial: ChemDiv No. D089-0563 50% (at 10 μM) 7.6 μM 2

Commercial: ChemDiv No. D089-0474 72% (at 10 μM) 95% viability (at 10μM) 3

Commercial: Otava Chemical No. 7018860541 52% (at 10 μM) 6.3 μM 4

Commercial: ChemDiv No. D089-0559 73% (at 10 μM) 75% viability (at 10μM) 5

Commercial: ChemBridge No. 9127171 89% (at 10 μM) 85% viability (at 10μM) 6

Commercial: Otava Chemical No. 7018860559 45% (at 10 μM) 4.9 μM 7

Commercial: Otava Chemical No. 7011860542 81% (at 10 μM) 7.2 μM 8

Commercial: Otava Chemical No. 7011860556 76% (at 10 μM) — 9

Commercial: ChemBridge No. 9139363 96% (at 10 μM) 100% viability (at 10μM) 10

Commercial: ChemBridge No. 6240697 37% (at 10 μM) 7.1 μM 11

Commercial: ChemBridge No. 6238398 69% (at 10 μM) 89% viability (at 10μM) 12

Commercial: ChemDiv No. D089-0350 43% (at 10 μM) 3.4 μM 13

Commercial: Otava Chemical No. 7018860553 12% (at 10 μM) 3.3 μM 14

New: 196D-12 106% (at 10 μM) 84% viability (at 10 μM) 15

New: 196D-15 80% (at 10 μM) 53% viability (at 10 μM) 16

New: 196D-16 112% (at 10 μM) 70% viability (at 10 μM) 17

Commercial: Otava Chemical No. 7018860464 68% (at 10 μM) >12 μM 18

Commercial: Otava Chemical No. 7018860259 6.5% (at 10 μM) 5.2 μM 19

Commercial: Otava Chemical No. 7018860288 0.56% (at 10 μM) 3.8 μM 20

Commercial: Otava Chemical No. 7018860345 1.4% (at 10 μM) 3.8 μM 21

New: 196D-44 74% (at 10 μM) >12 μM 22

New: 196D-48b 27% (at 10 μM) 100% viability (at 10 μM) 23

New: DC-34 0.44% (at 10 μM) 3.6 μM 24

New: 196D-49a 63% (at 6 μM) 7.8 μM 25

New: 196D-49b 32% (at 10 μM) 1.6 μM 26

New: DC-065B — 11.40 μM 27

New: DC-066B — 25.84 μM 28

New: DC-067B — 6.84 μM 29

New: DC-068B — 4.45 μM 30

New: DC-069B — 38.17 μM 31

New: DC-197 — 100% viability (at 10 μM) 32

New: DC-111B — 100% viability (at 10 μM) 33

New: DC-86B — 100% viability (at 10 μM) 34

New: DC-102B — 9.83 μM 35

New: DC-103B — 100% viability (at 10 μM)Additional analogs of Compound 1 are listed below:

Compound Structure 36

37

38

39

40

41

42

43

Additional multiple myeloma cell lines were assayed for cell viabilityin the presence of Compound 23, and the following IC50 values weredetermined:

Cell Line IC₅₀ (μM) L363 3.64 KMM1 4.13 KMS27 5.06 KMS12PE 5.33 ARD 5.75AMO1 5.92 JIM1 6.35

Example 3 Compounds 1 and 23 Reduce Growth of c-Myc Expressing Cells

This example illustrates that Compounds 1 and 23 can be used in inhibitthe growth of c-Myc expressing cancer cell lines in vitro. The NCI-60panel of cancer cell lines (see Shoemaker, Nat. Rev. Cancer, 6, 813-823,2006, and dtp.cancer.gov/discovery_development/nci-60/) was assayed forcell growth in the presence of 10 μM Compound 1 or Compound 23 for 48hours. At 48 hours, the assay was terminated by fixation with TCA anddetermination of relative cell counts by addition of Sulforhodamine B(SRB, see Alley et al., Cancer Research, 48: 589-601, 1988). Growthpercent was determined by comparison of SRB reading of a duplicate plateof cells taken at the time of compound addition to the SRB reading atthe termination of the treatment. The following table provides a summaryof the percent of growth of each cell line in the panel compared tocontrol (no treatment), as well as the mean delta and range of percentgrowth compared to control across the panel of cell lines for eachtreatment condition.

Compound 1 Compound 23 Cell Line Growth Percent Growth Percent LeukemiaCCRF-CEM 87.67 65.23 HL-60(TB) 92.74 83.01 K-562 75.93 29.63 MOLT-492.81 64.31 RPMI-8226 91.38 75.08 SR 83.12 −8.44 Non-Small Cell LungCancer A549/ATCC 85.87 61.22 EKVX 83.79 64.06 HOP-62 79.76 70.39 HOP-9255.89 34.88 NCI-H226 85.66 72.45 NCI-H23 90.23 69.93 NCI-H322M 97.5169.34 NCI-H460 80.25 51.53 NCI-H522 90.68 72.52 Colon Cancer COLO 205104.20 48.73 HCC-2998 116.36 11.22 HCT-116 64.67 32.68 HCT-15 83.4545.06 HT29 100.25 80.45 KM12 102.59 65.50 SW-620 85.97 67.44 CNS CancerSF-268 100.73 75.44 SF-295 85.34 48.84 SF-539 93.10 61.23 SNB-19 84.3569.94 SNB-75 78.34 50.57 U251 88.66 55.07 Melanoma LOX IMVI 88.87 39.15MALME-3M 107.60 49.92 M14 85.90 58.14 MDA-MB-435 95.94 58.22 SK-MEL-299.26 78.38 SK-MEL-28 96.60 76.90 SK-MEL-5 86.08 69.97 UACC-257 112.1182.00 UACC-62 102.82 67.27 Ovarian Cancer IGROV1 81.22 54.25 OVCAR-3101.03 62.02 OVCAR-4 82.06 58.58 OVCAR-5 95.63 85.79 OVCAR-8 89.96 66.34NCl/ADR-RES 89.34 53.65 SK-OV-3 100.26 76.35 Renal Cancer 786-0 84.6849.50 A498 64.12 50.48 ACHN 93.94 68.48 CAKI-1 75.13 50.46 RXF 393 81.3650.40 SN12C 84.31 66.72 TK-10 92.66 81.06 UO-31 62.74 40.69 ProstateCancer PC-3 79.90 45.70 DU-145 103.26 64.80 Breast Cancer MCF7 78.4148.09 MDA-MB-231/ATCC 79.39 48.96 HS 578T 88.40 65.50 BT-549 92.88 78.51T-47D 92.74 73.98 MDA-MB-468 73.62 5.74 Mean 88.39 58.56 Delta 32.5067.00 Range 60.47 94.23

The effect of Compound 23 on the expression of c-Myc protein in thehuman lung cancer cell lines H1299 and H157 was also assayed. H1299 andH157 cells were treated with 10 μM Compound 23 and c-Myc proteinexpression levels quantified by Western blot. In Compound 23-treatedH1299 cells, c-Myc protein expression levels were approximately 55%compared to untreated control cells. In Compound 23-treated H157 cells,c-Myc protein expression levels were approximately 74% compared tountreated control cells.

Example 4 Pharmacokinetic Evaluation of Compound 23

The blood plasma levels of Compound 23 after a single IP or IV injection(19 mg/kg) in female nude mice were determined by mass spectrometry (seeFIG. 12 and the following table).

Pharmacokinetic Parameters IV IP AUC_(0-24h) 491.729 552.478 AUC_(0-inf)493.342 570.140 T_(max) (h) 0.38 1.00 t_(1/2) (h) 2.25 6.50 Kel (1/h)0.277 0.277

Example 5 Pharmacodynamic Evaluation of Compound 23

The capacity of Compound 23 to inhibit c-MYC expression in tumor wasevaluated in nude mice bearing subcutaneous xenografts of the humanmultiple myeloma cell line L363. In this assay, tumors from twotumor-bearing mice were harvested 2 and 8 hours after a single dose ofCompound 23 at 19 mg/kg or 38 mg/kg (administered IP). By western blot,decreased c-MYC expression levels were observed in tumors at each timepoint compared to vehicle control tumors (FIG. 13).

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A method of treating cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, having astructure of:

and wherein the cancer is cancer with overexpression of the c-Myc gene.2. The method of claim 1, wherein administering the therapeuticallyeffective amount of the compound or the pharmaceutically acceptable saltthereof to the subject decreases growth and/or proliferation of thecancer.
 3. The method of claim 1, wherein the cancer is selected fromleukemia, non-small cell lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer,and multiple myeloma.
 4. A method of decreasing c-Myc expression in acell, comprising contacting the cell with an effective amount of acompound, or a pharmaceutically acceptable salt or ester thereof, havinga structure of:


5. The method of claim 4, wherein the cell is in vitro.
 6. The method ofclaim 4, wherein the cell is in vivo.
 7. The method of claim 4, whereindecreasing expression of c-Myc in the cell decreases growth and/orproliferation of the cell.
 8. The method of claim 4, wherein the cell isa cell with overexpression of the c-MYC gene.